Regulatory roles for small G proteins in the pancreatic {beta}-cell: lessons from models of impaired insulin secretion

Anjaneyulu Kowluru

Department of Pharmaceutical Sciences, Applebaum College of Pharmacy and Health Sciences and the {beta}-Cell Biochemistry Research Laboratory, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201


    ABSTRACT
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
Emerging evidence suggests that GTP-binding proteins (G proteins) play important regulatory roles in physiological insulin secretion from the islet {beta}-cell. Such conclusions were drawn primarily from experimental data derived through the use of specific inhibitors of G protein function. Data from gene depletion experiments appear to further substantiate key roles for these signaling proteins in the islet metabolism. The first part of this review will focus on findings supporting the hypothesis that activation of specific G proteins is essential for insulin secretion, including regulation of their function by posttranslational modifications at their COOH-terminal cysteines (e.g., isoprenylation). The second part will overview novel, non-receptor-dependent mechanism(s) whereby glucose might activate specific G proteins via protein histidine phosphorylation. The third section will review findings that appear to link abnormalities in the expression and/or functional activation of these key signaling proteins to impaired insulin secretion. It is hoped that this review will establish a basis for future research in this area of islet signal transduction, which presents a significant potential, not only in identifying key signaling proteins that are involved in physiological insulin secretion, but also in examining potential abnormalities in this signaling cascade that lead to islet dysfunction and onset of diabetes.

cytokines; posttranslational modifications; histidine phosphorylation; diabetes mellitus; pancreatic islet


GLUCOSE-INDUCED INSULIN SECRETION from pancreatic {beta}-cells is mediated largely via the generation of soluble second messengers, such as cyclic nucleotides, hydrolytic products of phospholipases (A2, C, and D), and adenine nucleotides (44, 48, 59, 60, 73, 81). However, the exact molecular and cellular mechanisms underlying glucose-stimulated insulin secretion remain only partially understood. It is widely accepted that, after its entry into the {beta}-cell (facilitated via the glucose-transporter protein GLUT2), glucose is metabolized with a resultant increase in the ATP/ADP ratio. Such an increase in the intracellular ATP results in the closure of ATP-sensitive K+ channels localized on the plasma membrane, as a consequence of which membrane depolarization occurs. This facilitates the influx of extracellular calcium through the voltage-sensitive calcium channels. Increase in intracellular calcium is known to be critical for the transport of insulin-containing secretory granules to the plasma membrane for fusion and release of insulin into circulation (73, 81).


    GTP-BINDING PROTEINS IN THE PANCREATIC {beta}-CELL AND THEIR REGULATION BY POSTTRANSLATIONAL MODIFICATIONS
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
In addition to regulation by adenine nucleotides of glucose-stimulated insulin secretion, earlier studies (59, 73, 81) have examined the contributory roles for guanine nucleotides (i.e., GTP) in physiological insulin secretion. For example, using selective inhibitors of the GTP biosynthetic pathway (e.g., mycophenolic acid), several studies have documented a permissive role for GTP in insulin secretion elicited by glucose (33, 68, 66). Although the precise mechanisms underlying the regulatory role(s) of GTP remain elusive, available evidence indicates that they might involve activation of one (or more) G proteins (44, 48, 85). Two major groups of G proteins have been identified in {beta}-cells (44, 48, 85). The first group consists of trimeric G proteins comprised of {alpha} (39–43 kDa)-, {beta} (35–37 kDa)-, and {gamma} (6–8 kDa)-subunits. These are involved in the coupling of various receptors to their intracellular effectors, such as adenylate cyclase, phosphodiesterase, or phospholipases (6, 17, 85). The second group of G proteins (which is the main focus of this review) is comprised of small-molecular-mass (20–25 kDa) monomeric G proteins, which are involved in protein sorting as well as trafficking of secretory vesicles (see Refs. 39, 44, 48 for reviews). A large body of evidence indicates that this family of G proteins undergoes posttranslational modifications, such as isoprenylation and carboxyl methylation, at their COOH-terminal cysteine residues (often referred to as the CAAX motif; 39, 44, 48).

The first of a four-step modification sequence (Fig. 1) includes incorporation of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety, which is derived from mevalonic acid (MVA), onto a cysteine residue toward the carboxyl terminus of the candidate G proteins. This is followed by proteolysis of several amino acids (up to a maximum of three). A carboxyl methylation step then modifies the newly exposed carboxylate anion of the cysteine. In some cases, the covalent addition of a long-chain fatty acid, typically palmitate, at cysteine residues, which are upstream to the CAAX motif, completes the cascade. Such modification(s) are thought to render the modified G proteins more hydrophobic and enable them to associate with membranes for interaction with their respective effectors (39, 44, 48, 92). Because the isoprenylation of G proteins occurs shortly after their synthesis, and because "half-lives" of prenylated proteins are rather long, this is not likely to be an acute regulatory step; however, in many cases, prenylation is necessary to allow candidate G proteins to intercalate into the relevant membrane compartment. In contrast, the methylation and acylation steps (Fig. 1) are subject to acute regulation at the level of the "on" steps (i.e., addition of methyl or acyl groups) as well as the "off" steps (i.e., deletion of methyl or acyl groups). The addition and removal of methyl groups are catalyzed by carboxyl methyl transferase and esterase, respectively. Likewise, addition and deletion of palmitoyl groups are facilitated by palmitoyl transferase and esterase, respectively (58). Studies from our laboratory (2, 3537, 39, 4153, 55, 65, 69) and those of others (34, 54, 56, 83) have demonstrated the requisite nature and roles of posttranslational modifications of these proteins in physiological insulin secretion. They are discussed in the following sections.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Posttranslational modifications of small G proteins. The first of the four-step reaction is incorporation of either a 15 (farnesyl)- or a 20 (geranylgeranyl)-carbon derivative of mevalonic acid (MVA) into the COOH-terminal cysteine via a thioether linkage. This reaction is catalyzed by either the farnesyl or geranylgeranyl transferases, respectively. After this, the three amino acids after the prenylated cysteine are removed by a protease of microsomal origin, thereby exposing the carboxylate anion. This site is then methylated by a carboxyl methyl transferase, which transfers a methyl group onto the carboxylate group using S-adenosyl methionine (SAM) as the methyl donor. We have shown that the carboxyl methylation of specific G proteins (e.g., Cdc42) increases their hydrophobicity and translocation to the membrane fraction (see text for additional details). In addition to these, certain G proteins (e.g., H-Ras) have also been shown to undergo palmitoylation at a cysteine residue, which is upstream to the prenylated cysteine. It is thought that palmitoylation provides a "firm" anchoring for the modified protein into the cell membrane for optimal interaction with its respective effector proteins. FPP, farnesyl pyrophosphate; FTase, farnesyl transferase; CMT, carboxyl methyl transferase; PMT, palmitoyl transferase.

 

Islet G Protein Prenylation and Insulin Secretion

Using generic as well as more specific inhibitors (see Table 1), numerous earlier studies have demonstrated critical regulatory roles for protein prenylation in physiological insulin secretion and identified some of these proteins as Cdc42, H-Ras, {gamma}-subunits of trimeric G proteins, and the nuclear lamin-B (2, and see Ref. 48 for a review). Needless to say, this list is only partial. Initial studies that examined possible roles of protein prenylation in islet function utilized statins (56, 69, 106), as they inhibit the synthesis of MVA, a precursor for the biosynthesis of isoprenoid derivatives (e.g., farnesyl or geranylgeranyl pyrophosphates), which are incorporated into respective proteins to complete the isoprenylation step (Fig. 1). Preincubation of isolated normal rat islets or clonal {beta}-cells with lovastatin has been shown to result in selective accumulation of non-prenylated proteins in the soluble fraction, with a concomitant decrease in their abundance in the membrane fraction. Under these conditions, lovastatin significantly inhibited glucose-stimulated insulin secretion from normal rat islets (69) (Fig. 2) and from bombesin- and vasopressin-mediated insulin secretion HIT-T15 cells (56). Even though the identity of all of the G proteins critical for this process has not been determined, indirect evidence suggests that Cdc42 might represent one such protein. For example, in transformed {beta}-cells, lovastatin reduces prenylation of Cdc42 and thereby impedes its complexing with a GDP-dissociation inhibitor (83). This, in turn, leads to its redistribution from membranes to cytosol, effects not seen with some other monomeric G proteins [e.g., Rho or ADP ribosylation factor (ARF)]. Together, data from these studies indicate that inhibition of protein prenylation in {beta}-cells results in selective accumulation of unprenylated G proteins in the soluble compartment, possibly interfering with the interaction of these proteins with their respective effector proteins, which may be required for nutrient-induced insulin secretion. Data from studies using more generic inhibitors of protein isoprenylation (e.g., limonene, perillic acid; see Table 1) were not very conclusive because of their nonspecific and cytotoxic effects on islet function (39, 56, 69).


View this table:
[in this window]
[in a new window]
 
Table 1. Known effects of inhibitors of posttranslational modifications of G proteins on insulin secretion

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Inhibitors of posttranslational modifications of small G proteins markedly reduce glucose-stimulated insulin secretion from normal rat islets. Effects of inhibitors of prenylation [e.g., lovastatin (LOVA); 15 µM], carboxyl methylation [e.g., acetyl farnesyl cysteine (AFC); 100 µM], or palmitoylation [e.g., cerulenin (CER); 134 µM] on glucose (16.7 mM)-stimulated (control) insulin secretion from normal rat islets are shown as indicated. Representative data from studies described in our earlier publication (69) were plotted in this figure. Data indicate a marked attenuation by inhibitors of all 3 classes of requisite modifications of small G proteins of glucose-stimulated insulin secretion from normal rat islets. *P < 0.001. (See Table 1 for a summary of observations from various laboratories on potential regulation by these modification steps of insulin secretion elicited by various insulin secretagogues in different insulin-secreting cell types.)

 

Recently, we synthesized a novel class of prodrug inhibitors, such as 3-allyl and vinyl-farnesols and 3-allyl and 3-vinyl geranylgeraniols, which inhibited (with a greater specificity) the protein farnesyl and geranylgeranyl transferases, respectively. These two classes of inhibitors significantly reduced glucose- and calcium-stimulated insulin secretion from {beta}-TC3 cells (2). The degree of inhibition was much greater than what was demonstrable in the presence of lovastatin in isolated rat islets, suggesting that they are much more site specific than the classical hydroxymethyl glutaryl-CoA reductase blockers (Table 1). Akin to lovastatin, allyl and vinyl farnesols and geranylgeraniols significantly influenced the subcellular distribution of small G proteins, as evidenced by a considerable degree of accumulation of the unprenylated proteins in the cytosolic fraction, with a concomitant decrease in their abundance in the membrane fraction (2). Together, these cited studies indicate that protein prenylation plays a significant regulatory role in physiological insulin secretion. It is also apparent that a substantial amount of work is still needed, especially in the area of identification of these prenylated proteins, as well as the prenylating enzymes (e.g., isoprenyl transferases). Recent evidence from our laboratory suggests immunological localization of farnesyl and geranylgeranyl transferases in insulin-secreting cells (40). As we have pointed out, even though protein prenylation is not acutely regulable, it seems to dictate the subsequent modification steps (e.g., carboxyl methylation) that are acutely regulated and to determine the functional status of a given G protein.

Islet G Protein Methylation and Insulin Secretion

Unlike protein prenylation, the carboxyl methylation of prenylated cysteine is acutely regulable, and both the methylating and demethylating enzymes have been characterized in mammalian cells, including the pancreatic {beta}-cell (39, 41, 55). The carboxyl methyl transferase catalyzes the incorporation of a methyl group onto the carboxylate anion of the prenylated cysteine via an ester linkage. It utilizes intracellular S-adenosyl methionine (SAM) as the methyl donor. Several studies, including our own, have identified carboxyl-methylated proteins in the pancreatic {beta}-cell. These include Cdc42, Rap1, Rac 1, H-Ras, the {gamma}-subunits of trimeric G proteins, and the nuclear lamin-B (35, 41, 49, 54, 95).

A previous study characterized the prenyl cysteine methyl transferase activity in insulin-secreting cells and normal rat islets (55). This activity was monitored by quantitating the degree of methylation of an artificial substrate [e.g., acetyl farnesyl cysteine (AFC)] with [3H]SAM as methyl donor. Subcellular fractionation studies revealed that this enzyme is localized in the plasma membrane and the endoplasmic reticular fractions. Even though several lines of experimental evidence indicate that the carboxyl methylation of specific G proteins (e.g., Cdc42 and Rap1) is stimulated by exogenous GTP (49, 54), we observed that exogenous GTP had no demonstrable effect on this enzyme, suggesting that this enzyme may be constitutively active within the {beta}-cell, and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyl transferase, as well as their active GTP-bound conformation (55). It may be germane to point out that, in addition to the carboxyl methylation at COOH-terminal cysteine, we reported methylation of COOH-terminal leucine, especially of the catalytic subunit of protein phosphatase 2A (PP2Ac) (51). Inhibitors of protein phosphatases, such as okadaic acid, inhibited the carboxyl methylation of PP2Ac. Data derived from the inhibitor experiments provide useful insights into the applicability of inhibitors of protein carboxyl methylation for study of putative roles of different proteins in cellular regulation. For example, AFC inhibits the methylation at a COOH-terminal cysteine, whereas okadaic acid specifically inhibits the carboxyl methylation of COOH-terminal leucine (41, 49, 51, 54).

Several earlier investigations have examined the relevance of prenyl cysteine carboxyl methylation in glucose-induced insulin secretion (49, 69) (see Fig. 2). For example, by use of rat islets and clonal {beta}-cells, glucose has been shown to stimulate the carboxyl methylation of Cdc42 and Rap1 in a transient manner. Stimulation of carboxyl methylation of these proteins was demonstrable within 15–30 s after exposure of cells to glucose (49). It was also shown that such an increase in the carboxyl methylation of these proteins was specifically blocked by AFC, because a structurally similar inactive analog of AFC, namely, acetyl geranylgeranyl cysteine (AGGC), was without any effect. Studies from Fleischer's group (Leiser et al., Ref. 54) have also utilized these specific probes to determine the relative contribution of Rap 1, another monomeric G protein, in glucose- and calcium-mediated insulin secretion. Follow-up studies from our laboratory have utilized similar experimental approaches and probes to decipher the roles of the carboxyl methylation of the {gamma}-subunits of trimeric G proteins in glucose-mediated insulin secretion (41).

Finally, by use of specific inhibitors of GTP biosynthesis [e.g., mycophenolic acid (MPA)], it was possible to establish a critical requirement for endogenous GTP in glucose-stimulated carboxyl methylation of specific G proteins and concomitant stimulation of insulin secretion from isolated rat islets (39, 41, 49). Depletion of endogenous GTP markedly reduced the ability of glucose to stimulate the carboxyl methylation of specific islet proteins (e.g., Cdc42, G{gamma}-subunits of trimeric G proteins) as well as insulin secretion, suggesting that endogenous GTP is essential for these signaling steps leading to insulin secretion (39, 41, 49). Such a formulation was further supported by additional observations indicating that provision of guanosine exogenously to GTP-depleted cells completely reversed the ability of glucose to activate the carboxyl methylation of these two proteins, as well as insulin secretion. The reversal effects appear to be specific for guanosine, since exogenous adenosine failed to reverse the inhibitory effects demonstrable after GTP depletion (39, 41, 49). These data indicate a clear dependence of endogenous GTP in physiological insulin secretion, presumably mediated by the activation of trimeric as well as monomeric G proteins. The reader is referred to Table 1 for a summary of findings from various laboratories on the effects of inhibitors of protein carboxyl methylation on insulin secretion from isolated {beta}-cells.

Islet G Protein Palmitoylation and Insulin Secretion

As indicated in Fig. 1, fatty acids (typically, palmitate) are incorporated posttranslationally into specific G proteins via a thioester linkage at cysteine residues upstream of the prenylated and methylated cysteine (48, 92, 103). This modification is thought to further facilitate the interaction of G proteins with their membrane-bound effectors. Several previous studies indicated that the {alpha}-subunits of trimeric G proteins may be acylated; this is regulated acutely in response to receptor activation, thereby controlling the subcellular distribution of these {alpha}-subunits (i.e., membrane vs. cytosolic). Receptor activation has also been shown to regulate protein deacylation (103). Cerulenin, a selective blocker of protein acylation, has been shown to reduce nutrient-induced insulin secretion from isolated rat islets (69) (Fig. 2); these data were further confirmed also in normal rat islets by Yajima et al. (109). Interestingly, cerulenin failed to inhibit insulin secretion facilitated by nonnutrient secretagogues, such as a membrane-depolarizing concentration of potassium, activators of protein kinase A, or mastoparan. Together, these data support a critical regulatory role for protein acylation steps in {beta}-cell function. It may be mentioned that the inhibitory effects of cerulenin (specifically, at higher concentrations and over longer periods of incubation) on protein acylation are rather nonspecific, because this probe can inhibit fatty acid, sterol, and protein synthesis. 2-Bromopalmitate has also been used to study the roles of protein acylation in cellular function (102). More specific cerulenin analogs have been reported recently (13) and await further investigations. Interestingly, experimental and structural data indicate that certain proteins, which undergo prenylation as well as carboxyl methylation (e.g., Cdc42 or {gamma}-subunits of trimeric G proteins), are not subject to fatty acylation (see Ref. 48 for a review). Therefore, it is likely that acylation of {alpha}-subunits of trimeric G proteins and/or other low-molecular-weight G proteins (e.g., Ras) may also be necessary for insulin secretion. Alternatively, other proteins involved in the exocytotic process, such as SNAP-25 (18, 97), may be critically acylated. Additional studies are needed to demonstrate conclusively a putative role(s) for fatty acylation, as well as the identity of candidate G proteins in physiological insulin secretion.

Use of Clostridial Toxins To Examine the Role of G Proteins in Insulin Secretion

Several lines of evidence suggest that clostridial toxins serve as extremely useful tools to study putative regulatory roles of the Rho subfamily of G proteins in cellular function (39, 42, 49, 86). These toxins specifically monoglucosylate and inactivate G proteins with reliable specificity (Table 2). For example, Clostridium difficile toxins A or B monoglucosylate (at threonine residues) Rho, Rac, and Cdc42 (but not Ras, Rab, or ARF) proteins; this modification impairs the function of these small G proteins. Clostridium sordellii lethal toxin monoglucosylates Rac, Rap, and Ras specifically, but not Cdc42, Rho, or Rab. In recent years, clostridial toxins have been used to seek further support for the above formulation that Rho proteins (e.g., Cdc42 and Rac) are involved in {beta}-cell signal transduction. Exposure of normal rat islets or clonal {beta}-cells to C. difficile toxin A or B significantly reduced glucose-induced insulin secretion. These data indicated that Rac, Cdc42, and Rho G proteins are involved in this phenomenon (42). Interestingly, C. sordellii toxin also reduced glucose-induced insulin secretion from these cells under similar experimental conditions, suggesting that Ras, Rap, and Rac are also involved in this phenomenon. C3 exoenzyme, which ADP ribosylates and inactivates Rho, failed to inhibit glucose-induced insulin secretion from these cells, suggesting that Rho may not be involved in this process (42). Together, these findings have led to the conclusion that Cdc42, Rap, Rac (all geranylgeranylated proteins), and Ras (a farnesylated protein) might be involved in physiological insulin secretion. These findings are compatible with our observations using allyl farnesols and geranylgeraniols (2).


View this table:
[in this window]
[in a new window]
 
Table 2. Specificity of bacterial toxins used for addressing the roles of small G proteins in stimulus-secretion coupling of the islet {beta}-cell

 

Use of Mastoparan to Examine the Role of G Proteins in Insulin Secretion

Mastoparan (Mas), a tetradecapeptide from wasp venom, has been shown to activate a wide variety of heterotrimeric as well as small G proteins, presumably by facilitating GTP/GDP exchange (21, 22). Several earlier studies have demonstrated that Mas stimulates insulin secretion from normal rat islets, human islets, and clonal {beta}-cells (see Table 3 for a summary of these studies). However, the precise loci for Mas regulation of insulin secretion remain less understood. Recent evidence from our laboratory suggested that Mas-induced insulin secretion from isolated {beta}-cells involves activation of Rac (3). Further experiments indicated that Mas activates Rac via GTP/GDP exchange but not via modulation of its isoprenylation. Transfection of dominant negative Rac (N17 Rac) markedly attenuated Mas-induced (3) or glucose- and forskolin-induced (57) insulin secretion from clonal {beta}-cell preparations, suggesting that Rac plays an important role in insulin secretion elicited by different secretagogues. Recent investigations by Daniel et al. (11) have also identified Cdc42 as one of the proteins involved in Mas-stimulated insulin secretion. Mas and Mas-17 (its inactive analog) are also proven to be as valuable probes in recent studies (Fig. 3), which addressed the insulin-secretory abnormalities in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes mellitus (NIDDM) (67). We reported that, whereas glucose- and potassium-induced insulin secretion was reduced significantly in islets from the GK rat, the Mas-induced insulin secretion remained unaltered in these islets. In GK islets, we also observed significant defects in the functional activation of nucleoside diphosphate kinase (NDPK), and on the basis of these data we proposed that the abnormalities in insulin secretion in the GK rat may lie at the level of an NDPK-mediated Mas-sensitive G protein (see the following sections for a summary of these and other related studies).


View this table:
[in this window]
[in a new window]
 
Table 3. Summary of data from earlier studies that used mastoparan to study stimulus-secretion coupling in the islet {beta}-cell

 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Structure-specific stimulation by mastoparan (Mas) of insulin secretion from isolated rat islets. Insulin release was measured from fresh, isolated normal rat islets in static incubation conditions at 3.3 mM glucose. Thirty micromoles each of Mas or Mas-17 (an inactive analog of Mas) were present during the 45-min incubation period, as indicated. Representative data from studies described in our earlier publication (43) were plotted. Data represent means ± SE from 3–5 determinations in each case. *P < 0.001 vs. control.

 

When the evidence just described is considered as a whole, it is evident that the Rho subfamily of G proteins play critical regulatory roles in physiological insulin secretion. However, it must be kept in mind that most, if not all, studies that were cited above (and in Tables 1 and 2) utilized chemical inhibitors of the requisite posttranslational modifications (e.g., statins) or bacterial toxins (e.g., clostridial toxins). Such approaches are often "questioned" for the nonspecific nature of the chemical inhibitors and toxins used to arrive at the respective conclusions. Although the degree of specificity of these probes was well studied and described, definitive support to the extant studies and further proof of potential regulatory roles for these G proteins in physiological insulin secretion must also be verified by gene depletion approaches. At the outset, at least three members of the Rho subfamily of G proteins, namely Cdc42, Rap1, and Rac1, must be given serious consideration for gene depletion studies to assess their contributory roles in physiological insulin secretion from the isolated {beta}-cell. This suggestion is based on the following experimental evidence. First, we (41, 49) and others (54) have demonstrated that Cdc42, Rac1, and Rap1 undergo glucose- and calcium-mediated carboxyl methylation and subsequent activation in normal rat islets and clonal {beta}-cells. Second, clostridial toxins, with defined specificity for inactivation of these proteins, markedly reduced glucose- and calcium-mediated insulin secretion (42). Third, novel geranylgeranyl transferase inhibitors, with the highest degree of specificity to inhibit these proteins, markedly reduced glucose-and calcium-induced insulin secretion from {beta}-cells (2). Taken together, these data assign major regulatory roles for Cdc42, Rac1, and Rap1 in physiological insulin secretion. More recent molecular biological data along these lines tend to further support a role for these G proteins in insulin secretion. Daniel et al. (11) reported a marked stimulation in Mas-induced insulin secretion in {beta}-cells after expression of Cdc42. Using a dominant negative mutant for Rac1 (N17 Rac1), we recently reported significant inhibition in Mas-induced (3) and glucose- and forskolin-induced (57) insulin secretion from isolated {beta}-cells. Although these data are encouraging, additional studies are needed to further verify the putative regulatory roles of these signaling proteins in insulin secretion, specifically via gene depletion approaches.

Together, on the basis of information reviewed above, it is clear that activation of certain G proteins, specifically those belonging to the Rho subfamily, is important for insulin secretion elicited by glucose and other secretagogues in the {beta}-cell. It is also becoming increasingly evident that abnormalities in the activation of specific G proteins could contribute to alterations in the insulin secretion demonstrable in models of impaired insulin secretion (see the following sections). The fundamental question of how glucose (and other insulin secretagogues) activate the islet endogenous G proteins still remains unanswered at this time. Along these lines, we (41, 42, 49) and others (54) have provided experimental evidence to indicate that glucose augments posttranslational modifications (e.g., carboxyl methylation) of specific G proteins (e.g., Cdc42 and Rap1) in a GTP-sensitive manner. In addition to these possibilities, and on the basis of more recent data obtained in our laboratory, I propose that activation of candidate G proteins by glucose may be mediated via the transphosphorylation of GDP bound to G proteins (inactive conformation) to their GTP-bound active conformation through the intermediacy of novel protein histidine kinases that we have recently identified in the islet {beta}-cell (see Table 4).


View this table:
[in this window]
[in a new window]
 
Table 4. Known histidine kinases, their potential phosphoprotein substrates, and their subcellular localization in insulin-secreting cells

 


    NOVEL REGULATORY MECHANISMS FOR THE ACTIVATION OF G PROTEINS IN THE ISLET {beta}-CELL: EVIDENCE FOR THE INVOLVEMENT OF PROTEIN HISTIDINE PHOSPHORYLATION
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
In most cells, the transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation of one (or more) G proteins and their effector systems (6, 17). The pancreatic {beta}-cell is unusual in that glucose, the major physiological agonist, lacks an extracellular receptor. Instead, events consequent to glucose metabolism promote insulin secretion via the generation and/or altered distribution of diffusible second messengers, such as ions, cyclic nucleotides, and biologically active lipids (44, 48, 59, 60, 73, 81). Changes in calcium concentration not only initiate insulin secretion but also regulate various enzymes, such as protein kinases, phosphodiesterases, adenylyl cyclases, and phospholipases, thereby facilitating insulin secretion. In addition to calcium-dependent protein kinase(s), several other kinases, including calmodulin-, cyclic nucleotide-, phospholipid-dependent protein kinases, tyrosine kinases, and mitogen-activated protein kinases have been described in {beta}-cells (see Ref. 27 for a review). The majority of these kinases mediate phosphorylation of endogenous {beta}-cell proteins using ATP as the phosphoryl donor. In addition, we (52) reported evidence for the localization of a novel protein kinase in {beta}-cells that selectively uses GTP as a phosphoryl donor and uniquely phosphorylates specific proteins (e.g., {beta}-subunit of trimeric G proteins) at histidine residues. We (52) further demonstrated that this phosphate, in turn, is transferred to free GDP (or GDP liganded to G proteins) to yield free GTP (or GTP bound to G proteins).

Protein Histidine Kinases

To date, the most phosphorylated amino acids identified include serine (P-Ser), threonine (P-Thr), and tyrosine (P-Tyr). Phosphoamino acids exhibit differential sensitivities to acidic and alkaline pH conditions (62). P-Ser and P-Thr, which form O-p (alcoholic O-monoester) linkages, are stable at acidic pH and are fairly unstable under alkaline conditions. P-Tyr, which forms O-p (phenolic O-monoester), is stable under acidic and alkaline conditions. Therefore, because of their stability under acidic conditions, P-Ser, P-Thr, and P-Tyr are readily identified after acid hydrolysis of phosphorylated proteins. However, acid-labile phosphoramidate linkage has been reported (62, 63) in histidine (P-His), arginine (P-Arg), and lysine (P-Lys). It is not surprising that very little information is available on the number of proteins with P-His, since its phosphate is rapidly lost during identification of phosphoamino acids under standard acid hydrolysis conditions or under conditions used for SDS-PAGE (52, 62, 63, 104). It is estimated that P-His may account for 6% of total protein phosphorylation in eukaryotes. In this context, it has also been shown that P-His undergoes rapid dephosphorylation in crude cellular extracts (28, 30), including pancreatic islet cell lysates, as we reported in Ref. 52.

Several recent studies have investigated protein histidine phosphorylation in multiple cell types. For example, Huang et al. (25) purified a monomeric histidine kinase from Saccharomyces cerevisiae with an apparent molecular mass of 32 kDa. This kinase exhibited specificity toward ATP (also GTP, but with minimal affinity) to phosphorylate histone-4. This enzyme required divalent cations for maximal activity; spermine or spermidine was ineffective. Motojima and Goto (70) reported histidine phosphorylation of a 36-kDa protein by a histidine kinase in liver extracts. They also reported localization of an okadaic acid-resistant phosphatase activity (with an apparent molecular mass of 45 kDa). Using an HPLC method, they demonstrated copurification of the kinase and p36 substrate at a 70- to 75-kDa size. These data indicate that the liver histidine kinase may be different from the yeast enzyme originally described by Huang et al. Along similar lines, Urushidani and Nagao (96) also reported autophosphorylation, at a histidine residue, of a 40-kDa protein localized in the membrane fraction derived from rabbit gastric mucosa. Sequence analyses data indicated that this protein might represent the {alpha}-subunit of an extramitochondrial isoform of succinyl-CoA synthetase (SCS) or its homolog. Autophosphorylation of this protein was stimulated by GDP, Ras (a small G protein), and myelin basic protein and was rapidly dephosphorylated in the presence of ATP, succinate, and CoA. Hegde and Das (19) showed that Ras stimulated the phosphorylation of a 36-kDa protein at a histidine residue in liver membranes. More recently, Besant and Attwood (5) purified and characterized a histone 4-phosphorylating histidine kinase activity from porcine thymus. This enzyme appears to have certain similarities with the yeast enzyme, including the molecular mass, which was estimated to be ~34–41 kDa. Together, these studies identified localization of a histidine-phosphorylating enzyme(s) that appears to be regulated under various experimental conditions (e.g., in the presence of Ras). The reader is referred to several recent reviews (1, 30, 71, 79, 89, 93) that describe potential regulatory roles of various histidine kinases in cellular regulation and function.

Using SDS-PAGE and the nitran filter paper assay, we (37) recently characterized a protein histidine kinase in the lysates of normal rat islets, human islets, and clonal {beta}-cell (HIT-T15 and INS-1) cell preparations. The {beta}-cell histidine kinase is sensitive to ATP as well as GTP, with an apparent molecular mass of 60–70 kDa. Noticeable similarities appear to exist between the {beta}-cell and the yeast histidine kinases. For example, both use ATP as well as GTP as phosphoryl donors, and both enzymes exhibit similar metal ion requirements and were resistant to polyamines. The principal difference appears to be the size of the enzyme. The {beta}-cell enzyme is ~60–70 kDa in size in contrast to the yeast enzyme, which has been shown to be ~32 kDa. On the basis of our additional observations in the {beta}-cell, we suggest that phosphohistidine phosphorylation may be important in insulin exocytosis from the {beta}-cell. In support of this formulation, we demonstrated (37) that the {beta}-cell histidine kinase is activated in a structure-specific manner by Mas. Mas or Mas-7, but not Mas-17 (an inactive analog), is a potent activator of insulin secretion (Table 2). We observed similar specificities for the activation by Mas analogs of histidine kinase activity, as well as the {beta}-subunit phosphorylation and insulin secretion, in rat islet homogenates (37). Although several previous studies, including our own (see Table 3), have demonstrated insulinotropic effects of Mas, our data suggest for the first time that Mas-mediated signaling events could include activation of protein histidine phosphorylation in the pancreatic {beta}-cell. Furthermore, these data establish a biochemical link between activation of histidine kinase and activation of phosphorylation of the {beta}-subunit through the use of Mas, a global G protein activator. Additional studies are needed to understand precisely the regulation of this enzyme by nutrient insulin secretagogues and G protein-coupled receptor agonists to conclusively establish a link between activation of G proteins (via activation of this or other related histidine kinases) and insulin secretion from isolated {beta}-cells. On the basis of our data on histidine kinase-mediated phosphorylation of the {beta}-subunit of trimeric G proteins, we propose a model for the activation of trimeric G proteins in the {beta}-cell involving protein histidine phosphorylation (Fig. 4). We propose that physiological insulin secretagogues (e.g., glucose) elicit effects on functional activation of specific G proteins via receptor-independent mechanisms. Our model predicts that glucose and other nutrient secretagogues stimulate histidine phosphorylation of specific "transmitter" proteins (e.g., the {beta}-subunit of trimeric G proteins) and that this phosphate, in turn, is transferred to a "receiver" protein, such as the {alpha}-subunit (in its GDP-bound inactive conformation) to yield its GTP-bound active conformation. In support of our hypothesis that cellular metabolism leads to rapid protein histidine phosphorylation, Crovello et al. (8) provided the first direct evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon activation. Using human platelets, they demonstrated transient phosphorylation of P-selectin at a histidine residue by thrombin or collagen. Although the activation mechanism proposed in Fig. 4 pertains to trimeric G proteins, it is also likely that similar activation mechanisms are operable in the context of small G proteins. This may be mediated via the NDPK-catalyzed reaction. In the following sections, we propose a model (Fig. 5) that predicts nutrient-mediated regulation of NDPK, which in turn generates GTP in the "vicinity" of candidate small G proteins necessary for their activation. Alternatively, NDPK could subserve the function of transphosphorylating the GDP bound to G proteins (i.e., their inactive conformation) to their GTP-bound, active conformation.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Proposed mechanism for receptor-independent activation of trimeric G proteins in the pancreatic {beta}-cell by glucose. Trimeric G proteins remain inactive when their {alpha}-subunit is bound to GDP. We propose that a histidine kinase phosphorylates the {beta}-subunit of trimeric G proteins at a histidine residue via a phosphoramidate linkage. This phosphate in turn is relayed to the GDP-bound {alpha}-subunit and transphorylates the GDP to GTP. Then, the {alpha}-subunit bound to GTP dissociates from the {beta}{gamma}-complex for regulation of its effector proteins. Ample experimental evidence identified multiple effector proteins for the {alpha}-subunits as well as the {beta}{gamma}-complex in several cellular systems. After hydrolysis of GTP by GTPase activity intrinsic to the {alpha}-subunit, {alpha}-GDP reassociates with the {beta}{gamma}-complex to complete one activation cycle. Not shown here is the possibility of nucleoside diphosphate kinase (NDPK) subserving the role of histidine kinase in mediating the phosphorylation of the {beta}-subunits (see text for additional details).

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Proposed mechanisms for glucose-stimulated activation of G proteins involving members of the histidine kinase family. We propose that, in addition to increasing GTP biosynthesis, glucose activates NDPK to facilitate transphosphorylation of GDP to GTP. Such an increase in GTP levels, specifically in the "vicinity" of candidate G proteins, results in activation of those G proteins, leading to stimulation of insulin secretion (left). On the basis of recent data (reviewed in the text), it is also likely that NDPK activation leads to direct activation of specific G proteins, which remain complexed with "activated" NDPK (middle). We propose that glucose also activates islet endogenous histidine kinase, which we have shown to phosphorylate the {beta}-subunit of trimeric G proteins (Fig. 2), thereby facilitating the activation of cognate trimeric G protein. Glucose could also mediate histidine phosphorylation of other proteins (e.g., ATP citrate lyase, aldolase, succinyl thiokinase) that are critical to glucose metabolism, thereby generating signals necessary for insulin secretion.

 

NDPK

The enzyme NDPK catalyzes the transfer of terminal phosphates from nucleoside triphosphates (e.g., ATP) to nucleoside diphosphates (e.g., GDP) to yield their respective nucleoside triphosphates (e.g., GTP). The transfer of terminal phosphates occurs by a two-step, ping-pong reaction involving the formation of a transient high-energy phosphoprotein intermediate form of NDPK, due to phosphorylation at a histidine residue, followed by transfer of that phosphate to a suitable acceptor (29). In addition to the generation of nucleoside triphosphates, NDPK has been implicated in the direct activation of certain G proteins as well as phosphorylation and/or regulation of several key enzymes of intermediary metabolism (e.g., ATP citrate lyase, aldolase, pyruvate kinase, glucose-6-phosphatase, and SCS) (15, 53, 89, 96, 99, 100).

Although multiple roles have been described for NDPK [the reader is referred to recent reviews on NDPK describing potential regulatory roles of this enzyme in regulation of cellular function (29, 89)], one of the unique roles of NDPK (in the context of this current review and {beta}-cell metabolism) is its ability to contribute toward the synthesis of GTP and the subsequent activation of specific G proteins. The latter is thought to occur via chaneling of GTP to the "vicinity" of candidate G proteins for their functional activation. It has also been shown that NDPK mediates transphosphorylation of GDP bound to G proteins (inactive conformation) to their GTP-bound (active conformation) of G proteins (43). Original studies from our laboratory (43) have characterized NDPK activity in normal rat and human islets as well as clonal {beta}-cell preparations. More recent studies (53) have identified at least three isoforms of NDPK in the pancreatic {beta}-cell. They include nm23-H1, a predominantly membrane-associated form of NDPK, and nm23-H2, with a membranous as well as soluble localization. In addition, a mitochondrial isoform of NDPK (nm23-H4) has been identified in the islet {beta}-cell (53). Potential roles of these isoforms and significance of their subcellular distribution have also been described in Ref. 53. On the basis of our current understanding of the biochemical properties and physiological regulation of this enzyme in the islet {beta}-cell, we propose a model for potential contributory roles of NDPK in glucose-stimulated insulin secretion, specifically at the level of activation of G proteins (Fig. 5). We propose that glucose-induced increases in the GTP/GDP ratio (as demonstrated earlier in Refs. 12 and 66) may in part be due to the activation of NDPK, which generates GTP via transphosphorylation of GDP from ATP. This increase in GTP concentrations favors either increase in GTP/GDP exchange on a relevant G protein[s] or chaneling of GTP to candidate G protein(s), culminating in their activation. In addition, it is likely that glucose also activates the histidine kinase (as described in the previous section), resulting in stimulation of the phosphorylation of key regulatory proteins, including the {beta}-subunits of trimeric G proteins at a histidine residue. Such a phosphate, in turn, is transferred to the GDP bound to the {alpha}-subunits of trimeric G proteins via the phospho-relay mechanism (52, 71, 79, 105) that is given in Fig. 4. We also propose that glucose-mediated activation of NDPK might result in histidine phosphorylation of other proteins, such as SCS, aldolase, and ATP-citrate lyase, which is required for their functional activation, and subsequent insulin secretion. For example, SCS catalyzes the substrate level phosphorylation of ADP or GDP. In the context of SCS regulation in the islet {beta}-cell, we have recently shown that the {alpha}-subunit of SCS undergoes phosphorylation at a histidine residue, which may be catalyzed by NDPK-mediated phosphotransfer mechanisms (36, 53). In support of this, we have demonstrated colocalization as a complex of mitochondrial NDPK and SCS in the {beta}-cell mitochondria. Using the mitochondrial extracts from clonal {beta}-cells (INS-1 and HIT-T15), we have been able to quantitate the formation of succinyl-CoA from succinate, CoA, and ATP or GTP. Furthermore, using immunological methods, we localized {alpha}- and {beta}-subunits of ATP-as well as GTP-sensitive isoforms of SCS in the {beta}-cell. In addition, using [{gamma}-32P]ATP as a phosphoryl donor, we observed that the {alpha}-subunit of SCS undergoes autophosphorylation at a histidine residue; coprovision of exogenous succinate and CoA resulted in pronounced dephosphorylation of the phosphorylated {alpha}-subunit of SCS. Taking these observations together, we provide evidence for the localization of two distinct activities of SCS in the {beta}-cell mitochondria. Whereas it is well established that ATP is critical for {beta}-cell metabolism, we propose that GTP generated by the activation of SCS, whose functional regulation is mediated via histidine phosphorylation, could promote key functional roles in the mitochondrial metabolism that lead to insulin secretion (36, 53).

As I review these cited studies, I think that the relay of high-energy phosphates as a consequence of protein histidine phosphorylation constitutes an important non-receptor-mediated activation of specific G proteins (and other proteins relevant to nutrient metabolism) by physiological stimuli such as glucose. Additional studies are required to substantiate such a hypothesis. In this context, two recent studies have provided additional support to our original formulation (52) for the non-receptor-dependent activation of G proteins involving protein histidine phosphorylation and high-energy phosphate transfer. First, Cuello et al. (9) reported activation of trimeric G proteins by a high-energy phosphate transfer from the histidine-phosphorylated NDPK to the {beta}-subunit of trimeric G proteins. Using bovine retinal and brain preparations, these investigators observed that the B isoform of NDPK forms complexes with the {beta}{gamma}-subunits of trimeric G proteins and contributes to the activation of the respective G protein by increasing the high-energy phosphate transfer from a transiently phosphorylated His266 in the {beta}-subunit to the GDP bound to the {alpha}-subunit, to yield an active conformation. In the second study, Hippe et al. (24) demonstrated the existence of a complex between NDPK (B isoform) and the {beta}{gamma}-complex of trimeric G proteins, and they implicated a role for NDPK in the phosphorylation of the {beta}-subunit, which is then transferred to the GDP bound to the {alpha}-subunit, resulting in its active, GTP-bound conformation. Interestingly, these findings are compatible with our recent observations on the existence of NDPK and succinyl thiokinase complexes in {beta}-cells (53), on the basis of which we proposed a role for NDPK in the functional regulation of succinyl thiokinase. It is likely that the mitochondrial NDPK might interact with other mitochondrial proteins as well. This is plausible, especially in light of recent observations of Srere and coworkers (87, 98) that clearly indicated the existence of complexes (appropriately termed "metabolons") of sequential metabolic enzymes involved in the tricarboxylic acid cycle. Together, it appears likely that the histidine kinase and various isoforms of NDPK that we characterized recently (Table 3) could subserve the function of histidine phosphorylation of key proteins (e.g., monomeric G proteins or subunits of trimeric G proteins), leading to the generation of appropriate signals necessary for physiological insulin secretion (37, 52, 53).

Several recent studies have identified additional roles for NDPK, such as its ability to interact with guanine nucleotide exchange factors for specific G proteins and subserve the function of activating specific GTPases (77, 78, 111). Although these regulatory mechanisms have not been fully studied in the islet {beta}-cell, we (95) and others (4) have obtained evidence to indicate localization of such factors (e.g., the guanine nucleotide exchange factor 1, or GRF1) in insulin-secreting cells. We (45) also described localization of similar exchange factors in normal islet and clonal {beta}-cells, which appear to be regulated by phospholipase-derived mediators of insulin secretion (e.g., arachidonic acid, lysophosphatidylcholine, and phosphatidic acid). In this context, we observed (unpublished observations) potential regulation of the islet NDPK activity by lipid messengers of insulin secretion (e.g., arachidonic acid). Although it seems likely, it remains to be seen whether nutrient-stimulated insulin secretion involves interplay between lipid messengers of insulin secretion, NDPK, guanine nucleotide exchange factors, and their effector G proteins within confines of a stimulated {beta}-cell. Furthermore, studies by Wagner and Vu (101) have identified roles for NDPK in the phosphorylation of farnesyl and geranylgeranyl triphosphates, which form precursors for G protein isoprenylation. In conclusion, a growing body of evidence is emerging to suggest critical regulatory roles for this enzyme, which was originally believed to play the role of a "house-keeping gene." On the basis of the above-mentioned reasons, it is logical to expect an increased interest in the area of putative regulatory roles of protein histidine phosphorylation in metabolic function and stimulus-secretion coupling, not only of the {beta}-cell but of other endocrine cells as well.


    ISLET G PROTEINS IN MODELS OF IMPAIRED INSULIN SECRETION
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
Recent evidence from multiple laboratories appears to suggest abnormalities in the expression and/or function of G proteins in animal and in vitro models of impaired insulin secretion. The majority of these studies were aimed at understanding the functional status of trimeric as well as monomeric G proteins. A relatively large body of evidence is emerging on alterations in the expression and function of G protein metabolism in islets derived from the GK rat, a widely accepted genetically determined rodent model for human type 2 diabetes. For example, we previously reported (67) that insulin secretion elicited in the presence of stimulatory concentrations of glucose, succinic acid methyl ester, or a depolarizing concentration of KCl was significantly impaired in GK rats. Interestingly, insulin secretion elicited by Mas was markedly increased above and beyond the stimulatory effects of this compound in control Wistar rat islets. We also demonstrated a significant reduction in the ATP-as well as GTP-sensitive phosphorylation and catalytic function of NDPK in islets derived from the GK rat. On the basis of these findings, we suggested that a defect in the late signaling steps in these islets, possibly occurring at the site of activation by NDPK of a Mas-sensitive G protein-dependent step, might contribute toward impaired insulin secretion in this animal model. More recent evidence from our laboratory (38) has also indicated significant defects in ATP-and GTP-sensitive histidine kinase activity in these islets, which we have implicated in the activation of specific G proteins (Figs. 4 and 5). Together, these data tend to support the viewpoint that abnormalities in protein histidine phosphorylation might lead to impaired insulin secretion.

Alterations in the expression and function of trimeric G proteins have also been reported in islets from the GK rat. Using immunohistochemical techniques, Frayon et al. (16) reported altered expression of adenylate cyclase isoforms and G{alpha}.olf in two models of NIDDM, namely, the GK rat and the neonatally treated streptozotocin (nSTZ)-induced diabetic rats. Interestingly, relative abundance of the adenylate cyclase II and adenylate cyclase III isoforms was clearly increased in both types of rats. The expression of G{alpha}.olf was increased in GK rat islets, whereas it was markedly attenuated in islets derived from the nSTZ rat. On the basis of these data, the authors concluded that alterations in the expression of G protein isotypes could contribute to the diabetic phenotype. Along similar lines, studies by Portela-Gomes and Abdel-Halim (80) indicated significantly higher expression of G{alpha}S and G{alpha}.olf and adenylate cyclase I and III isoforms in GK rat islets compared with control rat islets, suggesting possible alterations in the signaling mechanisms involving Gs proteins to adenylate cyclase isoforms in islets from the diabetic animals.

Yaekura et al. (107) provided additional insights into putative regulatory roles of small G proteins, specifically, Rab3A, in insulin secretion. They observed that Rab3A (a small G protein)-null mice developed fasting hyperglycemia and glucose intolerance. Insulin secretion in response to arginine was similar in control and Rab3A knockout mice, indicating a phenotype akin to insulin-secretory abnormalities demonstrable in type 2 diabetes. Despite no major differences in {beta}-cell mass and insulin production between the control and null groups, secretagogue-induced insulin secretion was impaired significantly in islets derived from Rab3A-null mice. Furthermore, glucose oxidation and glucose-induced increments in intracellular calcium concentrations were comparable in the two groups. On the basis of these data, the authors concluded that Rab3A plays a major role in glucose-induced insulin secretion by replenishing the readily releasable pool of insulinladen secretory granules.

Experiments by Srivastava et al. (88) in the Anx7 (a gene that encodes for a calcium-activated GTPase) knockout mouse have provided evidence into calcium signaling through inositol triphosphate stores in glucose-induced insulin secretion. These investigators demonstrated significant reduction in glucose-stimulated insulin secretion from the islets derived from the knockout mouse despite considerably high (8- to 10-fold) insulin content in mutants compared with their control counterparts. Even though the glucose-induced increases in intracellular calcium concentrations were comparable between control and knockout mice, the ability of inositol triphosphate-generating agonists to mobilize intracellular calcium was significantly attenuated in Anx 7(+/-) knockout mouse islets. These studies thus established a link between putative contributory roles for inositol triphosphate-mobilizable calcium stores in glucose-stimulated insulin secretion and its regulation by a calcium-activated GTPase in the islet {beta}-cell.

In summary, even though the list of studies that addressed the issue of possible contributory roles of G proteins in models of impaired secretion is somewhat short, it is my hope that this area will gain further recognition and momentum in the coming years, specifically with the availability of more advanced methodology, including the gene array technology. Together, on the basis of data derived from experiments involving specific inhibitors of G protein functions (e.g., inhibitors of posttranslational modification as well as clostridial toxins), gene depletion approaches, and transgenic animal models, it is reasonable to draw an overall conclusion that small G proteins play key regulatory roles in the signal transduction mechanisms leading to insulin secretion, and that abnormalities in the expression and/or functional activation of these signaling proteins lead to impairment in insulin secretion. In the following section, I will briefly review our current understanding of the roles of these signaling proteins in cytokine-mediated dysfunction and demise of the pancreatic {beta}-cell, which is a well-accepted model for insulin-dependent diabetes mellitus. Other aspects of putative roles of GTP and its binding proteins in {beta}-cell mitogenesis and survival have been reviewed in Ref. 65.


    G PROTEINS IN CYTOKINE-INDUCED {beta}-CELL DYSFUNCTION AND DEMISE
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
It is well established that insulin-dependent diabetes mellitus develops as a consequence of the selective destruction of insulin-secreting {beta}-cells, and it has also been proven beyond doubt that demise of the {beta}-cell is mediated by cytokines (e.g., IL-1{beta}) secreted by the infiltrating immune cells (10, 61, 64). Several lines of experimental evidence suggest that the demise of the pancreatic {beta}-cells due to immune attack could be due to the apoptotic and necrotic pathways. In the context of apoptosis, numerous studies have demonstrated the involvement of low-molecular-mass G proteins in multiple cell types (see Ref. 49 for a recent review). However, very little is known with respect to the regulatory roles of small G proteins in IL-1-induced {beta}-cell dysfunction and demise. In this context, we have recently begun to address the issue of small G proteins in cytokine-mediated dysfunction and demise of the islet {beta}-cell, and data along these lines of investigation provided convincing evidence to indicate that Ras plays a significant role in IL-1-mediated nitric oxide release from isolated rat islets and clonal {beta}-cell preparations (47, 94, 95). Again, as above, specific inhibitors of posttranslational modifications of G proteins, as well as bacterial toxins, were utilized to decipher the role of Ras in this phenomenon. Compatible with these observations are other reports that suggested key regulatory roles for GTP in the survival of the islet {beta}-cell (see Ref. 65 for a review). Together, these data clearly provide the initial evidence, in the context of the {beta}-cell, that GTP and G proteins play very important functional roles in the normal functioning of the islet, and that proapoptotic G proteins (e.g., Ras) play roles in the propagation of cellular events responsible for the cytokine-induced loss of {beta}-cell mass, leading to the onset of insulin-dependent diabetes mellitus (47, 94, 95). Clearly, this area is in its infancy, and additional studies are needed to identify these candidate pro-and antiapoptotic G proteins. This is an important area of investigation, since such data could provide valuable insights into the development of therapeutic intervention modalities for the prevention of loss of {beta}-cell mass.


    CONCLUSIONS AND FUTURE DIRECTIONS
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
From the discussion above, it is apparent that small-molecular-mass G proteins play key regulatory roles in the stimulus-secretion coupling of the islet {beta}-cell. These conclusions were reached on the basis of studies using mostly biochemical, physiological, and limited gene depletion approaches. We propose that glucose-mediated, receptor-independent activation of these G proteins requires the intermediacy of protein histidine phosphorylation and subsequent relay of the high-energy phosphate to GDP bound to G proteins to yield their respective GTP-bound active conformation. It also appears that alterations in the expression and/or functional activation of these proteins lead to impaired insulin secretion. Furthermore, specific G proteins (e.g., Ras) seem to play proapoptotic roles in the islet {beta}-cell after exposure to cytokines. It will be necessary to develop systems for the overexpression of G proteins or application of antisense approaches for specific G proteins (and their modifying enzymes), not only to deduce the physiological functions of these proteins in modulating insulin secretion but also to develop potential therapeutic approaches to states of perturbed metabolic status and insulin release. For these reasons, there appears to be an immediate need for the development of novel inhibitors of G protein functions, especially for those proteins that control and propagate signal transduction steps leading to the generation of nitric oxide, and consequently leading to the metabolic dysfunction and demise of the pancreatic {beta}-cell. In addition to these pharmacological probes, identification of candidate G proteins might help us in the development of novel bioengineered cell lines, which are resistant to immune attack, for the treatment of diabetes in humans (14, 72).


    DISCLOSURES
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 
My research work was funded by the Department of Veterans Affairs (Merit Review and the Research Enhancement Award program grants), National Institute of Diabetes and Digestive and Kidney Diseases (DK-56005), the American Diabetes Association, the Burroughs Wellcome Trust, and the Grodman Cure Foundation.


    ACKNOWLEDGMENTS
 
I thank the Medical Research Service of the Department of Veterans Affairs for the Research Career Scientist Award. I sincerely thank all of my former colleagues at the University of Wisconsin-Madison and my current associates at Wayne State University-Detroit who contributed to the work that I have described in this review.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Kowluru, Dept. of Pharmaceutical Sciences, 3601, Applebaum College of Pharmacy and Health Sciences, 259 Mack Ave., Wayne State Univ., Detroit, MI 48202 (E-mail: akowluru{at}med.wayne.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 GTP-BINDING PROTEINS IN THE...
 NOVEL REGULATORY MECHANISMS FOR...
 ISLET G PROTEINS IN...
 G PROTEINS IN CYTOKINE-INDUCED...
 CONCLUSIONS AND FUTURE...
 DISCLOSURES
 REFERENCES
 

  1. Alex LA and Simon MI. Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet 10: 133–138, 1994.[ISI][Medline]
  2. Amin R, Chen HQ, Tannous M, Gibbs R, and Kowluru A. Inhibition of glucose- and calcium-induced insulin secretion from {beta}TC3 cells by novel inhibitors of protein isoprenylation. J Pharmacol Exp Ther 303: 82–88, 2002.[Abstract/Free Full Text]
  3. Amin R, Chen HQ, Veluthakal R, Li J, Li G, and Kowluru A. Novel roles for Rac1 in mastoparan-induced insulin secretion. Diabetes 52, Suppl 1: 1604, 2003.
  4. Arava Y, Seger R, and Walker MD. GRF{beta}, a novel regulator of calcium signaling, is expressed in pancreatic {beta} cells and brain. J Biol Chem 274: 24449–24452, 1999.[Abstract/Free Full Text]
  5. Besant PG and Attwood PV. Detection of mammalian histone H4 kinase that has yeast histidine kinase-like enzymatic activity. Int J Biochem Cell Biol 32: 243–253, 2000.[ISI][Medline]
  6. Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell 71: 1069–1072, 1992.[ISI][Medline]
  7. Boitard C, Larger E, Timsit J, Sempe P, and Bach JF. IDD: an islet or an immune disease? Diabetologia 37, Suppl 2: S90–S98, 1994.[ISI][Medline]
  8. Crovello CS, Furie BC, and Furie B. Histidine phosphorylation of P-selectin upon stimulation of human platelets: a novel pathway for activation-dependent signal transduction. Cell 82: 279–286, 1995.[ISI][Medline]
  9. Cuello F, Schulz RA, Heemeyer F, Meyer HE, Lutz S, Jakobs KH, Niroomand F, and Wieland T. Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Complex formation of NDPK B with Gbeta gamma dimers and phosphorylation of His-266 IN Gbeta. J Biol Chem 278: 7220–7226, 2003.[Abstract/Free Full Text]
  10. Cunningham JM and Green IC. Cytokines, nitric oxide and insulin secreting cells. Growth Regul 4: 173–180, 1994.[ISI][Medline]
  11. Daniel S, Noda M, Cerione RA, and Sharp GW. A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. Biochemistry 41: 9663–9671, 2002.[ISI][Medline]
  12. Detimary P, Van den Berghe G, and Henquin JC. Concentration dependence and time course of the effects of glucose on adenine and guanine nucleotides in mouse pancreatic islets. J Biol Chem 271: 20559–20565, 1996.[Abstract/Free Full Text]
  13. De Vos ML, Lawrence DS, and Smith CD. Cellular pharmacology of cerulenin analogs that inhibit protein palmitoylation. Biochem Pharmacol 62: 985–995, 2001.[ISI][Medline]
  14. Efrat S. Cell replacement therapy for type 1 diabetes. Trends Mol Med 8: 334–340, 2002.[ISI][Medline]
  15. Feldman F and Butler LG. Detection and characterization of the phosphorylated form of microsomal glucose-6-phosphatase. Biochem Biophys Res Commun 36: 119–125, 1969.[ISI][Medline]
  16. Frayon S, Pessah M, Giroix MH, Mercan D, Boissard C, Malaisse WJ, Portha B, and Garel JM. G{alpha}.olf identification by RT-PCR in purified normal pancreatic B cells and in islets from rat models of non-insulin-dependent diabetes. Biochem Biophys Res Commun 254: 269–272, 1999.[ISI][Medline]
  17. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615–649, 1987.[ISI][Medline]
  18. Gonzalo S and Linder ME. SNAP-25 palmitoylation and plasma membrane targeting require a functional secretory pathway. Mol Biol Cell 9: 585–597, 1998.[Abstract/Free Full Text]
  19. Hegde AN and Das MR. Ras proteins enhance the phosphorylation of a 38 kDa protein (P38) in liver plasma membrane. FEBS Lett 217: 74–80, 1987.[ISI][Medline]
  20. Hertelendy ZI, Patel DG, and Knittel JJ. Pancreastatin inhibits insulin secretion in RINm5F cells through obstruction of G-protein mediated, calcium-directed exocytosis. Cell Calcium 19: 125–132, 1996.[ISI][Medline]
  21. Higashijima T, Burnier J, and Ross EM. Regulation of Gi and Go by mastoparan, related amphiphilic peptides and hydrophobic amines. J Biol Chem 265: 14176–14181, 1990.[Abstract/Free Full Text]
  22. Higashijima T, Uzu S, Nakajima T, and Ross EM. Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G-proteins). J Biol Chem 263: 6491–6494, 1988.[Abstract/Free Full Text]
  23. Hillaire-Buys D, Mousli M, Landry Y, Bockaert J, Fehrentsz JA, Carrette J, and Rouot B. Insulin releasing effects of mastoparan and amphiphilic substance P receptor antagonists on RINm5F insulinoma cells. Mol Cell Biochem 109: 133–138, 1992.[ISI][Medline]
  24. Hippe HJ, Lutz S, Curllo F, Knorr K, Vogt A, Jakobs KH, Wieland T, and Niroomand F. Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B Gbetagamma complex in H10 cells. J Biol Chem 278: 7227–7233, 2003.[Abstract/Free Full Text]
  25. Huang JM, Wei YF, Kim YH, Osterberg L, and Matthews HR. Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases. J Biol Chem 266: 9023–9031, 1991.[Abstract/Free Full Text]
  26. Jones PM, Mann FM, Persaud SJ, and Wheeler-Jones CP. Mastoparan stimulates insulin secretion from pancreatic betacells by effects at a late stage in the secretory pathway. Mol Cell Endocrinol 94: 97–103, 1993.[ISI][Medline]
  27. Jones PM and Persaud SJ. Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic {beta} cells. Endocrine Rev 19: 429–461, 1998.[Abstract/Free Full Text]
  28. Kim Y, Huang J, Cohen P, and Matthews H. Protein phosphatases 1, 2A, and 2C are protein histidine phosphatases. J Biol Chem 268: 18513–18518, 1993.[Abstract/Free Full Text]
  29. Kimura N, Shimada N, Fukuda M, Ishijima Y, Miyazaki H, Ishii A, Takagi Y, and Ishikawa N. Regulation of cellular functions by nucleoside diphosphate kinases in mammals. J Bioenerg Biomemb 32: 309–315, 2000.[ISI][Medline]
  30. Klumpp S and Krieglstein J. Phosphorylation and dephosphorylation of histidine residues in proteins. Eur J Biochem 269: 1067–1071, 2002.[Abstract/Free Full Text]
  31. Komatsu M, Aizawa T, Yokokawa N, Sato Y, Okada N, Takasu N, and Yamada T. Mastoparan-induced hormone release from rat pancreatic islets. Endocrinology 130: 221–228, 1992.[Abstract]
  32. Komatsu M, McDermott AM, Gillison SL, and Sharp GW. Mastoparan stimulates exocytosis at a Ca (2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line. J Biol Chem 268: 23297–23306, 1993.[Abstract/Free Full Text]
  33. Komatsu M, Noda M, and Sharp GW. Nutrient augmentation of calcium-dependent and calcium-independent pathways in stimulus-coupling to insulin secretion can be distinguished by their guanosine triphosphate requirements: studies on rat pancreatic islets. Endocrinology 139: 1172–1183, 1998.[Abstract/Free Full Text]
  34. Konrad RJ, Young RA, Record RD, Smith RM, Butkerait P, Manning D, Jarett L, and Wolf BA. The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis. J Biol Chem 270: 12869–12876, 1995.[Abstract/Free Full Text]
  35. Kowluru A. Evidence for the carboxyl methylation of nuclear lamin-B in the pancreatic {beta} cell. Biochem Biophys Res Commun 268: 249–254, 2000.[ISI][Medline]
  36. Kowluru A. Adenine and guanine nucleotide-specific succinyl-CoA synthetases in the clonal beta-cell mitochondria: implications in the beta-cell high-energy phosphate metabolism in relation to physiological insulin secretion. Diabetologia 44: 89–94, 2001.[ISI][Medline]
  37. Kowluru A. Identification and characterization of a novel protein histidine kinase in the islet {beta} cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion. Biochem Pharmacol 63: 2091–2100, 2002.[ISI][Medline]
  38. Kowluru A. Defective protein histidine phosphorylation in islets from the Goto-Kakizaki diabetic rat. Am J Physiol Endocrinol Metab 285: E498–E503, 2003.[Abstract/Free Full Text]
  39. Kowluru A and Amin R. Inhibitors of posttranslational modifications of G-proteins as probes to study the pancreatic {beta} cell function: potential therapeutic implications. Curr Drug Targets Immune Endocr Metabol Disord 2: 129–139, 2002.[Medline]
  40. Kowluru A, Chen HQ, and Tannous M. Novel roles for Rho subfamily of GTP-binding proteins in succinate-induced insulin secretion from {beta} TC3 cells. Endocr Res 2003. In press.
  41. Kowluru A, Li G, Rabaglia ME, Segu VB, Hofmann F, Aktories K, and Metz SA. Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells. Biochem Pharmacol 54: 1097–1108, 1997.[ISI][Medline]
  42. Kowluru A, Li G, and Metz SA. Glucose activates the carboxyl methylation of {gamma} subunits of trimeric GTP-binding proteins in pancreatic {beta} cells. J Clin Invest 100: 596–610, 1997.
  43. Kowluru A and Metz SA. Characterization of nucleoside diphosphokinase activity in human and rodent pancreatic {beta} cells: evidence for its role in the formation of guanosine triphosphate, a permissive factor for nutrient-induced insulin secretion. Biochemistry 33: 12495–12503, 1994.[ISI][Medline]
  44. Kowluru A and Metz SA. GTP and its binding proteins in the regulation of insulin exocytosis. In: Molecular Biology of Diabetes, edited by Draznin B and LeRoith D. Totowa, NJ: Humana, 1994, p. 249–283.
  45. Kowluru A and Metz SA. Regulation of guanine-nucleotide binding proteins in islet subcellular fractions by phospholipase-derived lipid mediators of insulin secretion. Biochim Biophys Acta 1222: 360–368, 1994.[ISI][Medline]
  46. Kowluru A and Metz SA. Stimulation by prostaglandin E2 of a high-affinity GTPase in the secretory granules of normal rat and human pancreatic islets. Biochem J 297: 399–406, 1994.[ISI][Medline]
  47. Kowluru A and Morgan NG. GTP-binding proteins in cell survival and demise: the emerging picture in the pancreatic {beta} cell. Biochem Phramacol 63: 1027–1035, 2002.
  48. Kowluru A, Robertson RP, and Metz SA. GTP-binding proteins in the regulation of pancreatic {beta} cell function. In: Diabetes Mellitus. A Fundamental and Clinical Text, edited by LeRoith D, Taylor SI, and Olefsky JM. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 78–94.
  49. Kowluru A, Seavey SE, Li G, Sorenson RL, Weinhaus AJ, Nesher R, Rabaglia ME, Vadakekalam J, and Metz SA. Glucose- and GTP-dependent stimulation of the carboxyl methylation of Cdc42 in rodent and human pancreatic islets and pure {beta} cells. J Clin Invest 98: 540–555, 1996.[Abstract/Free Full Text]
  50. Kowluru A, Seavey SE, Rabaglia ME, and Metz SA. Non-specific stimulatory effects of mastoparan on pancreatic islet nucleoside diphosphate kinase activity: dissociation from insulin secretion. Biochem Pharmacol 49: 263–266, 1995.[ISI][Medline]
  51. Kowluru A, Seavey SE, Rabaglia ME, Nesher R, and Metz SA. Carboxyl methylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion. Endocrinology 137: 2315–2323, 1996.[Abstract]
  52. Kowluru A, Seavey SE, Rhodes CJ, and Metz SA. A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells. Biochem J 313: 97–107, 1996.[ISI][Medline]
  53. Kowluru A, Tannous M, and Chen HQ. Localization and characterization of the mitochiondrial isoform of the nucleoside diphosphate kinase in the pancreatic {beta} cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase. Arch Biochem Biophys 398: 160–169, 2002.[ISI][Medline]
  54. Leiser M, Efrat S, and Fleischer N. Evidence that Rap1 carboxyl-methylation is involved in regulated insulin secretion. Endocrinology 136: 2521–2530, 1995.[Abstract]
  55. Li G, Kowluru A, and Metz SA. Characterization of prenylcysteine methyltransferase in insulin-secreting cells. Biochem J 316: 345–351, 1996.[ISI][Medline]
  56. Li G, Regazzi R, Roche E, and Wollheim CB. Blockade of mevalonate roduction by lovastatin attenuates bombesin and vasopressin potentiation of nutrient-induced insulin secretion from HIT-T15 cells. Biochem J 289: 379–385, 1993.[ISI][Medline]
  57. Li J, Luo R, Kowluru A, and Li G. Involvement of Rac1, a small G-protein, in islet {beta} cell growth and insulin secretion. Diabetes 52, Suppl 1: 1616, 2003.
  58. Linder ME and Deschenes RJ. New insights into the mechanisms of protein palmitoylation. Biochemistry 42: 4311–4320, 2003.[ISI][Medline]
  59. MacDonald MJ. Elusive proximal signals of beta-cells for insulin secretion. Diabetes 39: 1461–1466, 1990.[Abstract]
  60. Malaisse WJ. Hormonal and environmental modification of islet activity. In: Handbook of Physiology. Endocrine Pancreas. Bethesda, MD: Am. Physiol. Soc., 1972, vol. I, sect. 7, chapt. 14, p. 237–260.
  61. Mandrup-Poulsen T. The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia 39: 1005–1029, 1996.[Medline]
  62. Matthews HR. Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen activated protein kinase cascade. Pharmocol Ther 67: 323–350, 1995.
  63. Matthews HR and Chan K. Protein histidine kinase. Meth Mol Biol 124: 171–182, 2001.[Medline]
  64. McDaniel ML, Kwon G, Hill JR, Marshall CA, and Corbett JA. Cytokines and nitric oxide in islet inflammation and diabetes. Proc Soc Exp Biol Med 11: 24–32, 1996.
  65. Metz SA and Kowluru A. Inosine monophosphate dehydrogenase: a molecular switch integrating pleiotropic GTP-dependent {beta} cell functions. Proc Assoc Am Physicians 111: 335–346, 1999.[ISI][Medline]
  66. Metz SA, Meredith M, Rabaglia ME, and Kowluru A. Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets. J Clin Invest 92: 872–882, 1993.[ISI][Medline]
  67. Metz SA, Meredith M, Vadakekalam J, Rabaglia ME, and Kowluru A. A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats. Diabetes 48: 1754–1762, 1999.[Abstract]
  68. Metz SA, Rabaglia ME, and Pintar TJ. Selective inhibitors of GTP-synthesis impede exocytotic insulin release from intact rat islets. J Biol Chem 267: 12517–12527, 1992.[Abstract/Free Full Text]
  69. Metz SA, Rabaglia ME, Stock JB, and Kowluru A. Modulation of insulin secretion from normal rat islets by inhibitors of the posttranslational modifications of GTP-binding proteins. Biochem J 295: 31–40, 1993.[ISI][Medline]
  70. Motojima K and Goto S. Histidyl phosphorylation and dephosphorylation of P36 in liver extracts. J Biol Chem 269: 9030–9037, 1994.[Abstract/Free Full Text]
  71. Nederkoorn PHJ, Timmerman H, Timms D, Wilkinson AJ, Kelly DR, Broadley KJ, and Davies RH. Stepwise phosphorylation mechanisms and signal transmission within a ligand-receptor-G{alpha}{beta}{gamma}-protein complex. J Mol Struct 452: 25–47, 1998.
  72. Newgard CB, Clark S, BeltrandelRio H, Hohmeier HE, Quaade C, and Normington K. Engineered cell lines for insulin replacement in diabetes: current status and future prospects. Diabetologia 40, Suppl 2: S42–S47, 1997.[ISI][Medline]
  73. Newgard CB and McGarry JD. Metabolic coupling factors in pancreatic {beta} cell signal transduction. Ann Rev Biochem 64: 689–719, 1995.[ISI][Medline]
  74. Ohara-Imaizumi M, Nakamichi Y, Ozawa S, Katsuta H, Ishida H, and Nagamatsu S. Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action. Biochem Biophys Res Commun 289: 1025–1030, 2001.[ISI][Medline]
  75. Ohtsuki K, Ikeuchi T, and Yokoyama M. Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLaS3 cells. Biochim Biophys Acta 882: 322–330, 1986.[ISI][Medline]
  76. Ostenson CG, Zaitsev S, Berggren PO, Efendic S, Langel U, and Bartfai T. Galparan: a powerful insulin-releasing chimeric peptide acting at a novel site. Endocrinology 138: 3308–33013, 1997.[Abstract/Free Full Text]
  77. Otsuki Y, Tanaka M, Yoshi S, Kawazoe N, Nakaya K, and Sigimura H. Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1. Proc Natl Acad Sci USA 98: 4385–4390, 2001.[Abstract/Free Full Text]
  78. Palacios F, Schweitzer JK, Boshans RL, and D'Souza-Schorey C. ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly. Nat Cell Biol 4: 929–936, 2002.[ISI][Medline]
  79. Piacentini L and Niroomand F. Phosphotransfer reactions as a means of G protein activation. Mol Cell Biochem 157: 59–63, 1996.[ISI][Medline]
  80. Portela-Gomes GM and Abdel-Halim SM. Overexpression of Gs proteins and adenylyl cyclase in normal and diabetic islets. Pancreas 25: 176–181, 2002.[ISI][Medline]
  81. Prentki M and Matschinsky FM. Calcium, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev 67: 1223–1226, 1987.
  82. Rabuzzo AM, Buscema M, Caltabiano V, Anello M, Degano C, Patane G, Vigneri R, and Purrello F. Interleukin 1{beta} inhibition of insulin release in rat pancreatic islets: possible involvement of G-proteins in the signal transduction pathway. Diabetologia 38: 779–784, 1995.[ISI][Medline]
  83. Regazzi R, Kikuchi A, Takai Y, and Wollheim CB. The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J Biol Chem 267: 17512–17519, 1992.[Abstract/Free Full Text]
  84. Regazzi R, Sasaki T, Takahashi K, Jonas JC, Volker C, Stock JB, Takai Y, and Wollheim CB. Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells: comparison with the effects of Rab3AL peptide. Biochim Biophys Acta 1268: 269–278, 1995.[ISI][Medline]
  85. Robertson RP, Seaquist ER, and Walseth TF. G-proteins and modulation of insulin secretion. Diabetes 40: 1–6, 1991.[Abstract]
  86. Schmidt M, Rumenapp U, Bienek C, Keller J, von Eichel-Streiber C., and Jakobs KH. Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins. J Biol Chem 271: 2422–2466, 1996.[Abstract/Free Full Text]
  87. Srere P. Complexities of metabolic regulation. Trends Biochem Sci 19: 519–520, 1994.[ISI][Medline]
  88. Srivastava M, Atwater I, Glasman M, Leighton X, Goping G, Caohuy H, Miller G, Pichel J, Westphal H, Mears D, Rojas E, and Pollard HB. Defects in inositol 1,4,5-triphosphate receptor expression, Ca2+ signaling, and insulin secretion in the anx 7(+/-) knockout mouse. Proc Natl Acad Sci USA 96: 13783–13788, 1999.[Abstract/Free Full Text]
  89. Steeg PS, Palmieri D, Ouatas T, and Salerno M. Histidine kinases and histidine phosphorylated proteins in mammalian cell biology, signal transduction and cancer. Cancer Lett 190: 1–12, 2003.[ISI][Medline]
  90. Straub SG, James RF, Dunne MJ, and Sharp GW. Glucose augmentation of mastoparan-stimulated insulin secretion in rat and human pancreatic islets. Diabetes 47: 1053–1057, 1998.[Abstract]
  91. Straub SG, Yajima H, Komatsu M, Aizawa T, and Sharp GWG. The effects of cerulenin, an inhibitor of protein acylation, on the two phases of glucose-stimulated insulin secretion. Diabetes, Suppl 1: S91–S95, 2002.
  92. Takai Y, Sasaki T, and Matozaki T. Small GTP-binding proteins. Physiol Rev 81: 153–208, 2001.[Abstract/Free Full Text]
  93. Tan E, Besant PG, and Attwood PV. Mammalian histidine kinases: do they REALLY exist? Biochemistry 41: 3843–3851, 2002.[ISI][Medline]
  94. Tannous M, Amin R, Popoff MR, Fiorentini C, and Kowluru A. Positive modulation by Ras of interleukin-1betamediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells. Biochem Pharmacol 62: 1459–1468, 2001.[ISI][Medline]
  95. Tannous M, Veluthakal R, Amin R, and Kowluru A. IL-1{beta}-induced nitric oxide release from insulin-secreting {beta} cells: further evidence for the involvement of GTP-binding proteins. Diabet Metab 6: 3S78–3S84, 2002.
  96. Urushidani T and Nagao T. Calcium-dependent membrane bound protein fraction from rabbit gastric mucosa contains a protein whose histidyl residue is phosphorylated. Biochim Biophys Acta 1356: 71–83, 1997.[ISI][Medline]
  97. Veit M. Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin. Biochem J 345: 145–151, 2000.[ISI][Medline]
  98. Velot C, Mixon MB, Teige M, and Srere PA. Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. Biochemistry 36: 14271–14276, 1997.[ISI][Medline]
  99. Wagner PD, Steeg PS, and Vu ND. Two component kinaselike activity of nm23 correlates with its motility suppressing activity. Proc Natl Acad Sci USA 94: 9000–9005, 1997.[Abstract/Free Full Text]
  100. Wagner PD and Vu ND. Phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase. J Biol Chem 270: 21758–21764, 1995.[Abstract/Free Full Text]
  101. Wagner PD and Vu ND. Phosphorylation of geranyl and farnesyl pyrophosphates by nm23 proteins/nucleoside diphosphate kinases. J Biol Chem 275: 35570–35576, 2000.[Abstract/Free Full Text]
  102. Webb Y, Hermida-Matsumoto L, and Resh MD. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275: 261–270, 2000.[Abstract/Free Full Text]
  103. Wedegaertner PB, Wilson PT, and Bourne HR. Lipid modifications of trimeric G-proteins. J Biol Chem 270: 503–506, 1995.[Free Full Text]
  104. Wei YF and Matthews HR. A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation. Anal Biochem 190: 188–192, 1990.[ISI][Medline]
  105. Wieland T, Nunberg B, Ulibarri I, Kaldenberg-Stasch S, Schultz G, and Jakobs KH. Guanine nucleotide specific phosphate transfer by guanine-binding regulatory protein {beta} subunits. J Biol Chem 268: 18111–18118, 1993.[Abstract/Free Full Text]
  106. Yada T, Nakata M, Shiraishi T, and Kakei M. Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic calcium signaling and insulin secretion due to blockade of L-type calcium channels in rat islet {beta} cells. Br J Pharmacol 126: 1205–1213, 1999.[Abstract/Free Full Text]
  107. Yaekura K, Julyan R, Wickteed BL, Hays LB, Alarcon C, Sommers S, Poitout V, Baskin DG, Wang Y, Philipson LH, and Rhodes CJ. Insulin secretory deficiency and glucose intolerance in Rab3A null mice. J Biol Chem 278: 9715–9721, 2003.[Abstract/Free Full Text]
  108. Yaguchi H, Ohkura N, Tsukada T, and Yamaguchi K. Menin, the multiple endocrine neoplasia type 1 gene product, exhibits GTP-hydrolyzing activity in the presence of the tumor metastasis suppressor nm23. J Biol Chem 277: 197–204, 2002.
  109. Yajima H, Komatsu M, Yamada S, Straub SG, Kaneko T, Sato Y, Yamauchi K, Hashizume K, Sharp GW, and Aizawa T. Cerulenin, an inhibitor of protein acylation, selectively attenuates nutrient stimulation of insulin release: a study in rat pancreatic islets. Diabetes 49: 712–717, 2000.[Abstract]
  110. Yokokawa N, Komatsu M, Takeda T, Aizawa T, and Yamada T. Mastoparan, a wasp venom, stimulates insulin release by pancreatic islets through pertussis toxin-sensitive GTP-binding protein. Biochem Biophys Res Commun 158: 712–716, 1989.[ISI][Medline]
  111. Zhu J, Tseng YH, Kantor JD, Rhodes CJ, Zetter BR, Moyers JS, and Kahn CR. Interaction of the Ras-related proteins associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation. Proc Natl Acad Sci USA 96: 14911–14988, 1999.[Abstract/Free Full Text]