Department of Pharmaceutical Sciences, Wayne State University, Detroit
48202, and -Cell Biochemistry Research Laboratory, John D. Dingell
Veterans Affairs Medical Center, Detroit, Michigan 48201
Submitted 21 March 2003 ; accepted in final form 30 April 2003
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ABSTRACT |
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pancreatic islet; nucleoside diphosphate kinase; GTP-binding proteins; insulin secretion
Several studies have appeared in recent years on the localization,
characterization, and regulation of protein histidine kinases in multiple cell
types (2,
11,
28,
30,
39,
42). Wei and Matthews
(42) first reported a filter
paper-based protein kinase assay that selectively quantitated acid-labile and
alkali-stable phosphorylation reactions. Utilizing this assay, they were able
to purify and characterize a protein histidine kinase from Saccharomyces
cerevisiae by use of histone 4 as the substrate and demonstrated that the
yeast histidine kinase activity selectively phosphorylated the histidine
residue at the 75th position, but not the 18th residues on histone 4
(42). Along these lines, we
(18,
21) recently identified
several proteins that underwent histidine phosphorylation in islet
-cells. Some of these include the nucleoside diphosphate (NDP) kinase,
the
-subunit of trimeric G proteins
(22), and the succinyl
thiokinase (STK; Ref. 13). We
reported (18,
21) that the NDP kinase
undergoes autophosphorylation at a histidine residue, which in turn is
transferred to the NDPs to yield nucleotide triphosphates (NTPs). It has also
been proposed that NDP kinase mediates the transphosphorylation of GDP bound
to the G proteins (inactive conformation) to yield their respective GTP-bound
(active) conformation (12,
18,
37). We also reported that,
unlike the NDP kinase, the
-subunit of trimeric G proteins does not
undergo autophosphorylation but requires a membrane-associated factor or
kinase for its phosphorylation at a histidine residue
(14,
22). Furthermore, recent
studies from our laboratory
(13,
23) have localized in the
mitochondrial fraction from isolated
-cells a novel isoform of NDP
kinase, which appears to form a complex with the mitochondrial STK, possibly
mediating its functional regulation. Using histone 4 as the substrate, we
(14) recently characterized a
novel protein histidine kinase activity in normal rat islets, human islets,
and clonal
-cell preparations. This novel histidine kinase, with an
apparent molecular mass of 60-70 kDa, utilized either ATP or GTP as phosphoryl
donors. We also observed that mastoparan, a global stimulator of G proteins
and insulin secretion from isolated rat islets and clonal
-cells, but
not mastoparan-17, its inactive analog, stimulated this histidine kinase
activity in isolated
-cells
(14). Together, these data
suggested a critical role for protein histidine phosphorylation in the
-cell stimulus-secretion coupling.
Because we showed earlier that islet endogenous G proteins relevant to insulin secretion are activated by histidine phosphorylation, we undertook the present study to quantitate the degree of protein histidine phosphorylation in islets derived from the Goto-Kakizaki (GK) rat, a model for non-isulin-dependent diabetes mellitus (NIDDM) (29) to determine whether insulin-secretory abnormalities demonstrable in these cells relate to alterations in protein histidine phosphorylation. We provide evidence for a significant defect in the histidine phosphorylation of NDP kinase as well the histone 4-phosphorylating histidine kinase in islets derived from the GK rat. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.
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MATERIALS AND METHODS |
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Isolation of islets from control and diabetic rats. GK rats were generously provided by Dr. Robert Farese (Tampa, FL). They were provided to our laboratory (at the Veterans Affairs Medical Center, Madison, WI) at the age of 8 wk and were then housed at our animal care facility. Offspring were allowed to reach the ages of 8-14 wk, when they were studied in parallel with age- and sex-matched Wistar rats (Harlan, Indianapolis, IN). As indicated in Ref. 29, islets were isolated by collagenase digestion from control (Wistar) and diabetic (GK) rats. In brief, pancreases were inflated with collagenase solution (1 and 2 U/ml for Wistar and GK rats, respectively) in Hanks' balanced salt solution supplemented with 1% fetal bovine serum. After digestion with collagenase, islets were washed twice, passed through mesh (92-µm pore size), and then purified on a Ficoll gradient. Islets were then hand picked twice under stereomicroscopic control to exclude extraislet debris. Also, as indicated in our earlier studies (29), we made certain to isolate islets with grossly normal size and appearance to minimize any extrinsic artifacts of the isolation procedure and abnormal pancreatic morphology. Islets from control Wistar and diabetic GK rats were homogenized in a buffer consisting of 230 mM mannitol, 70 mM sucrose, and 5 mM HEPES buffer, pH 7.4, containing 1 mM EGTA, 1 mM DTT, and 2.5 µg/ml each of leupeptin and pepstatin.
Quantitation of histidine phosphorylation of NDP kinase in lysates from
control and diabetic rat islets. The phosphorylation reaction was carried
out (100 µl total volume), in a buffer consisting of 50 mM Tris ·
HCl, pH 7.4, 2 mM DTT, islet protein (30 µg), and
[-32P]ATP or [
-32P]GTP (1 µCi/tube) at
37°C for 2 min. It was terminated by the addition of Laemmli stop
solution. Because the phosphohistidine of NDP kinase is heat sensitive
(18,
21), the samples were
incubated with the sample buffer at room temperature for 30 min before
SDS-PAGE. Moreover, it has been shown
(18,
22) that typical fixation
conditions used for SDS gels (methanol, acetic acid, and water medium) ablates
phosphate labeling from [32P]phosphohistidine. Therefore, to verify
that our gel fixation conditions did not underestimate NDP kinase
phosphoenzyme formation, gels were fixed in some experiments in 50 mM sodium
phosphate buffer, pH 8.0, containing 18.5% formaldehyde for 1.5 h, as
described by Wei and Matthews
(42); these results were
compared with those obtained using gels fixed in methanol (40%) and acetic
acid (7%) medium. We observed no significant differences in the residual
labeling on NDP kinase between these two conditions (additional data not
shown). Therefore, after separation of proteins by SDS-PAGE, gels were fixed
in methanol-acetic acid medium for 1.5 h and dried at room temperature, as
described by us earlier (14,
18,
22). Labeled proteins were
identified by autoradiography. Molecular weights of labeled proteins were
determined using prestained molecular weight standards. Labeling intensity of
the proteins was quantitated by scanning individual lanes with a Zeineh Video
Laser Densitometer (Biomed Instruments, Fullerton, CA) that was interfaced to
an IBM computer equipped with software to calculate the individual peak
areas.
Quantitation of histidine kinase activity in lysates from control and
diabetic rat islets. This quantitation was done according to the method
we recently described (14). In
brief, the assay mixture (100 µl total volume) consisted of 0.6 mg/ml
histone 4, 0.2 mM [-32P]ATP or [
-32P]GTP,
15 mM magnesium chloride, 50 mM Tris · HCl, pH 7.5, and islet lysates,
as indicated (see legend to Fig.
1). The reaction was carried out at 37°C for 5 min and was
terminated by incubating the mixture in 0.5 N NaOH at 60°C for 30 min. The
base-treated reaction mixture was transferred directly to the Nytran filter
papers previously soaked overnight in 1 mM ATP, pH 9.0, at room temperature
and air dried. The filter papers were then transferred to a beaker with 200 ml
of 10 mM sodium pyrophosphate at pH 9.0 and gently stirred at room temperature
for 30 min to remove unreacted ATP or GTP. The filters were air-dried under an
infrared lamp, and the radioactivity associated with the filters was
quantitated by scintillation spectrometry. The degree of histidine
phosphorylation of endogenous proteins (no added histone 4) or exogenously
added histone 4 was expressed as picomoles of 32P incorporated per
minute per milligram of islet protein
(14).
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Other methods. The protein concentration in the samples was assayed according to the method of Bradford, using serum albumin as standard as we described (13, 15, 18, 20-22). The statistical significance of the differences between control and diabetic animal groups was determined by Student's t-test. P values <0.05 were considered significant.
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RESULTS |
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Figure 1 represents an
autoradiogram demonstrating the autophosphorylation of NDP kinase in control
(Wistar) and diabetic (GK) rat islets carried out using either
[-32P]ATP (A) or [
-32P]GTP
(B) as phosphoryl donors. These data indicate a substantial reduction
in NDP kinase phosphorylation in islets derived from the GK rat in the
presence of either of the substrates. Densitometric quantitation of the
labeled bands (Fig.
1C) indicated a >50% reduction in the
autophosphorylation of NDP kinase. These data are compatible with our recent
findings of a significant attenuation (-40%) in the catalytic activity of NDP
kinase in islets derived from the GK rats compared with their control
counterparts (29).
In addition to NDP kinase, we reported
(14) localization of a novel
histidine kinase activity in isolated rat islets and clonal -cells and
implicated this enzyme in the activation of islet G proteins and insulin
secretion. Herein, we measured such a histidine kinase activity, using a
Nitran filter paper assay, in homogenates derived from the control and GK rat
islets and in the absence or presence of histone 4 as the substrate. Data in
Fig. 2A indicate a
marked reduction (>90%) in the degree of endogenous protein histidine
phosphorylation (4.62 pmol 32P
incorporated·min-1·mg
protein-1 in control rat islets vs. 0.40 pmol
32P incorporated·min-1· mg
protein-1 in GK rat islets) when
[
-32P]ATP was used as the phosphoryl donor (see
MATERIALS AND METHODS for additional details). Furthermore, under
similar experimental conditions, we observed a substantial degree of
inhibition (-64%) of histidine phosphorylation of exogenously added histone 4
in the homogenates of diabetic GK rat islets compared with those from the
control Wistar rats (7 pmol 32P
incorporated·min-1·mg
protein-1 in Wistar rat islets vs. 2.5 pmol
32P incorporated·min-1·mg
protein-1 in GK rat islets). Although the
[
-32P]GTP-dependent phosphorylation of endogenous proteins
was reduced modestly, but significantly, the histone 4 phosphorylation was
markedly attenuated (-64%) in GK rat islets compared with the control Wistar
rat islets. The corresponding values for degree of endogenous protein
histidine phosphorylation were 1.12 pmol 32P
incorporated·min-1·mg
protein-1 in control rat islets vs. 0.8 pmol
32P incorporated·min-1·mg
protein-1 in GK rat islets. However, a nearly identical
degree of reduction was demonstrable in GTP-dependent phosphorylation of
histone 4 in GK rat islets (i.e., 1.84 pmol 32P
incorporated·min-1·mg
protein-1 in control rat islets vs. 0.66 32P
incorporated·min-1·mg
protein-1) in GK rat islets. Together, these data (Figs.
1 and
2) suggest significant
alterations in the protein histidine phosphorylation in the diabetic rat
islets.
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DISCUSSION |
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In the present studies, pancreatic islets were isolated from overtly diabetic, but nonobese, GK rats, a genetic model for NIDDM in humans and studied ex vivo compared with islets derived from age- and sex-matched control Wistar rats. However, we cannot exclude some contribution to the islet dysfunction by the antecedent, ambient diabetic milieu in vivo, since we have not attempted to restore normoglycemia in vivo or in cultured islets in vitro for prolonged periods at normal glucose concentration. Our previous data and the existing body of evidence (Ref. 29 and references therein) argue against the possibility that some degree of glucotoxicity or lipotoxicity might contribute to the attenuated histidine phosphorylation. Thus it seems likely that the defect in protein histidine phosphorylation that we report herein is intrinsic to the GK islet.
The possibility that a decreased -cell mass or decreased
-cell
protein could contribute to the observed defects in histidine phosphorylation
needs to be addressed as well in the context of our current findings. Earlier
studies have reported significant reductions in the DNA content or
-cell
mass in GK islets (31,
32), although others have
failed to see any differences in
-cell mass between control and GK rat
islets (8,
35). Recent data from our
laboratory have indicated no major differences in insulin content, protein
content, and the stereomicroscopic morphology between the Wistar and GK rat
islets (29). Furthermore, the
degree of reduction in ATP- or GTP-sensitive NDP kinase phosphorylation and
histidine kinase activity were significantly lower in GK rat islets even after
normalization of these values to equal protein (present studies). Together,
these points argue against a selective reduction in
-cell mass as the
(sole) islet lesion in the GK rat.
Emerging evidence indicates that several proteins of carbohydrate
metabolism undergo phosphorylation at histidine residues; these include
glucose-6-phosphatase (7),
ATP-citrate lyase (41), and
aldolase (40). Recent studies
from our laboratory (18,
21) have identified at least
three proteins that underwent histidine phosphorylation. These include NDP
kinase, the -subunit of trimeric G proteins
(22), and the mitochondrial
STK (13). In our present
studies, we observed that the ATP- and GTP-sensitive autophosphorylation of
the NDP kinase activity was significantly reduced in GK rat islets, data
compatible with marked reductions in NDP kinase catalytic activity from these
cells (29). In this context,
we have reported localization of at least three forms of NDP kinase in
isolated
-cells (23).
The nm-23 H1 (NDP kinase A) was found to be localized predominantly in the
soluble fraction, in contrast to nm23-H2 (NDP kinase B), which was found to be
localized in the soluble as well as the membranous fractions. Furthermore, a
third form of NDP kinase (i.e., nm23-H4) was found to be localized exclusively
in the mitochondrial fraction. Because in the present studies we utilized
total cell lysates instead of individual subcellular fractions, it is not
possible to identify conclusively which of these isoforms is defective in the
diabetic islet.
In the present study, we also observed significant reduction in ATP- and
GTP-sensitive histidine kinase activity in islets derived from the GK rat. We
have implicated this enzyme in the functional activation of G proteins in the
islet -cell (14). We
observed that, in addition to histone 4-phosphorylating activity, histidine
phosphorylation of endogenous proteins was also decreased. This could
potentially represent histidine phosphorylation of several islet endogenous
proteins, including NDP kinase, the mitochondrial STK, and the
-subunit
of trimeric G proteins that we have identified as proteins undergoing
phosphorylation at histidine residues (see above). On the basis of the data
presented in Fig. 1, one of the
potential proteins might represent the NDP kinase. Our unpublished evidence
along these lines indicates no major differences in the histidine
phosphorylation of the
-subunit. Additional studies are needed to
determine the identity of those proteins whose histidine phosphorylation is
significantly impaired in the GK islet.
In addition to the functional regulation of several enzymes, such as STK,
ATP-citrate lyase, glucose-6-phosphatase, aldolase, etc.
(7,
40,
41), recent evidence
implicates protein histidine phosphorylation in the activation of G proteins.
For example, several earlier studies, including our own (Ref.
14 and references therein)
demonstrated direct stimulatory effects of mastoparan, a tetradecapeptide from
wasp venom, on G protein function and insulin secretion in normal rat islets,
human islets, and clonal -cells. Recently, we
(14) also demonstrated that
mastoparan, but not its inactive analog mastoparan-17, markedly stimulated the
histone 4-phosphorylating histidine kinase activity in lysates derived from
normal rat islets and clonal
-cells. These data indicate a possible
regulatory role for this novel histidine kinase in the activation of G
proteins, presumably at the level of their conversion from a GDP-bound,
inactive form of the G proteins to its GTP-bound, active form. In this
context, independent studies from our laboratory in isolated
-cells
(22) and by Wieland et al.
(43) in HL-60 cells have
demonstrated that the
-subunit of trimeric G proteins undergoes
phosphorylation at a histidine residue and that phosphate, in turn, is
transferred to the GDP-bound
-subunit (inactive conformation) to yield
its GTP-bound, active conformation. We have also provided evidence
(14,
22) to suggest that, unlike
NDP kinase, which undergoes autophosphorylation at a histidine residue
(Fig. 1), the
-subunit
requires the intermediacy of a histidine kinase to catalyze its
phosphorylation at a histidine residue. These observations of novel activation
mechanisms of trimeric G proteins via subunit phosphorylation of
-subunits were confirmed recently by several studies
(5,
9,
33).
Together, it appears that protein histidine phosphorylation plays major
regulatory roles in metabolic regulation in multiple cell types, including the
islet -cell. Therefore, it is likely that a marked attenuation in the
histidine kinase activity demonstrable in diabetic rat islets (present study)
could result in the reduction in the activation of specific G proteins, which
we (1,
13,
15,
16,
19,
20) and others
(24,
25,
28) have shown to be essential
for insulin secretion. Our current data also provide further evidence to
suggest that abnormalities in insulin secretion demonstrable in GK rat islets
may be due, in part, to decreased histidine phosphorylation of specific islet
proteins; compatible with this formulation are our data in GK rats that
indicate a possible defect in the activation by NDP kinase of a
mastoparan-sensitive G protein step in the exocytotic secretion of insulin in
GK rats (29). Additional
studies are needed to precisely identify the phosphoprotein substrates for the
histidine kinase, whose phosphorylation may be relevant to insulin secretion.
Studies are also needed to identify the candidate G proteins (trimeric as well
as monomeric) whose activation is under the fine control of NDP kinase and
histidine kinase activity that we proposed to be essential for the exocytotic
secretion of insulin.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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A significant portion of this work was carried out during the author's stay at the Veterans Affairs Medical Center in Madison, WI, and the University of Wisconsin School of Medicine-Madison.
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FOOTNOTES |
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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.
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REFERENCES |
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