THEMES
G Protein-Coupled Receptors in Gastrointestinal Physiology
V. Gastrointestinal peptide signaling through tyrosine phosphorylation of focal adhesion proteins*

Enrique Rozengurt

Department of Medicine, Division of Digestive Diseases, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095

    ABSTRACT
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Abstract
Introduction
References

Gastrointestinal (GI) peptides (also referred to as neuropeptides or regulatory peptides), including the mammalian bombesin-like peptides gastrin and CCK, elicit the synthesis of classic second messengers (e.g., Ca2+, diacylglycerol, and cAMP) and the consequent stimulation of serine/threonine protein kinase cascades. An emerging theme in signal transduction is that these agonists also induce rapid and coordinate tyrosine phosphorylation of a set of focal adhesion proteins, including the nonreceptor tyrosine kinase p125fak and the adaptor proteins p130cas and paxillin. GI peptide-mediated induction of tyrosine phosphorylation of these focal adhesion proteins is critically dependent on the integrity of the actin cytoskeleton and on functional Rho. The purpose of this article is to review recent advances in unraveling this novel tyrosine kinase pathway(s), because it appears to play a fundamental role in the mediation of important biological effects induced by GI peptides, including cell migration and proliferation.

bombesin; p125 focal adhesion kinase; p130 c-Crk-associated substrate; Rho

    INTRODUCTION
Top
Abstract
Introduction
References

GASTROINTESTINAL (GI) peptides (also referred to as neuropeptides or regulatory peptides), including the mammalian bombesin-like peptides gastrin and CCK, play critical roles in the integration of exocrine and endocrine secretory and smooth muscle contractile functions in the GI tract. Produced by neural or endocrine cells, these signaling peptides reach their targets as neurotransmitters, paracrine regulators, or systemic hormones. The discovery that neuropeptides can also act as potent cellular growth factors identified a novel role for these informational molecules and provided fundamental evidence for establishing that G protein-coupled receptors (GPCR) also regulate cell proliferation (26).

It is increasingly recognized that the mitogenic effects of neuropeptides are relevant for normal and abnormal biological processes, such as development and neoplastic transformation. For example, multiple neuropeptides, including those of the bombesin/gastrin-releasing peptide (GRP) family, are implicated in the autocrine growth of human cancers, particularly small cell lung carcinoma (26). Consequently, the intracellular signal transduction pathways that mediate the biological effects induced by these multifunctional agonists are attracting major interest, because they may pinpoint potential targets for novel therapeutic interventions.

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of serine/threonine protein phosphorylation cascades is a well-known early response to GI peptides (6, 25, 26, 29, 32, 35). A key reaction in this process is the phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-biphosphate to produce two second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its intracellular receptor, a ligand-gated Ca2+ channel located in the endoplasmic reticulum membrane, and triggers the release of Ca2+ from internal stores. DAG directly activates the classic and novel isoforms of protein kinase C (PKC), which catalyze rapid phosphorylation of cytosolic and membrane-bound proteins. Further downstream, GI peptides trigger specific protein kinase cascades including Raf/MEK/ERK, rapamycin-sensitive p70S6K, and protein kinase D, leading to increased expression of immediate early response genes (e.g., c-fos, c-jun, c-myc), regulation of cell cycle events, and subsequent reinitiation of cell proliferation (29, 32, 35).

An emerging theme in the elucidation of the signal transduction pathways activated by regulatory peptides is that, in addition to eliciting the synthesis of classic second messengers (e.g., Ca2+, DAG, and cAMP) and the consequent stimulation of serine/threonine protein kinase cascades, these agonists also induce tyrosine phosphorylation of multiple proteins. The purpose of this article is to review recent advances in unraveling this novel tyrosine kinase pathway(s), because it appears to play a fundamental role in the biological effects induced by GI peptides.

    TYROSINE PHOSPHORYLATION OF FOCAL ADHESION KINASE (P125FAK), PAXILLIN, AND P130CAS

Bombesin and other GPCR agonists, including bradykinin, endothelin, and vasopressin, have been shown to stimulate a rapid increase in the tyrosine phosphorylation of multiple proteins, including heterogeneous bands of 110-130 and 70-80 kDa in Swiss 3T3 cells, a useful model system to elucidate signal transduction pathways in the action of regulatory peptides (reviewed in Ref. 25). Neuropeptide stimulation of these cells also increases tyrosine phosphorylation in cell-free preparations. These initial findings were surprising, because they demonstrated that activation of GPCRs, which are characterized by seven putative transmembrane alpha -helices, also induces tyrosine phosphorylation of multiple proteins in their target cells, apparently through stimulation of protein tyrosine kinase (PTK) activity.

Subsequent studies have identified p125fak, a nonreceptor PTK (12, 22), as one of the prominent tyrosine- phosphorylated bands in Swiss 3T3 cells stimulated by the GPCR agonists bombesin/GRP, bradykinin, endothelin, and vasopressin (30, 33). These agonists induce p125fak tyrosine phosphorylation in Swiss 3T3 cells at subnanomolar concentrations that closely parallel those necessary for mitogenic stimulation (30). Gastrin, CCK, and neuromedin B also lead to increased tyrosine phosphorylation of focal adhesion proteins in other model systems. The rapidity of neuropeptide-stimulated tyrosine phosphorylation (detectable within seconds) is consistent with the activation of a pathway that leads from GPCRs to p125fak.

p125fak and a second PTK [called proline-rich tyrosine kinase 2 (Pyk2), cell adhesion kinase-beta , related adhesion focal tyrosine kinase, or Ca2+-dependent PTK] comprise a new family of PTKs. Whereas p125fak is expressed in many cells and tissues, Pyk2 is found predominantly in brain and neural cells (19). Both proteins are structurally distinct nonreceptor PTKs characterized by a centrally located catalytic domain flanked by the NH2- and COOH-terminal noncatalytic domain of ~400 residues that do not contain Src homology 2 and 3 (SH2 and SH3) domains. The COOH-terminal region of p125fak contains a stretch of 159 amino acids, known as the focal adhesion targeting sequence, which is essential for focal adhesion localization and also mediates association of p125fak with other signal transduction and cytoskeletal proteins, including p130cas and paxillin (see below). Several studies have demonstrated that the major site of p125fak autophosphorylation (i.e., Tyr397) is a high-affinity binding site for the SH2 domain of members of the Src kinase family (reviewed in Refs. 12, 22). Complex formation between p125fak and Src results in the tyrosine phosphorylation of p125fak at additional sites that either upregulate its activity or provide docking sites for other signaling proteins.

The focal adhesion proteins paxillin and p130cas are potential downstream targets for p125fak and function as adaptors in signal transduction. Paxillin contains multiple domains that are thought to function in protein-protein interactions, including a proline-rich motif near the NH2 terminus that could bind SH3 domains, a region that interacts with p125fak and vinculin, and four regions homologous to LIM domains in the COOH-terminal portion of the molecule (2). p130cas contains an SH3 domain and a cluster of 15 potential SH2 domain-binding sites, of which 9 are sequences (YDV/TP) that correspond with high-affinity sites for the SH2 domain of the protooncogene Crk. p130cas also possesses binding sites for the SH2 domain of Src and proline-rich regions that could bind SH3 domain proteins (13). This suggests that tyrosine phosphorylated p130cas may serve to promote the assembly of multiple SH2-containing molecules.

Neuropeptides strikingly increase the tyrosine phosphorylation of paxillin and p130cas and promote the formation of a molecular complex between p130cas and the protooncogene c-Crk, an SH2 and SH3 domain-containing adaptor protein (5, 34). The complex between p130cas and c-Crk may be important in regulating the subcellular distribution of c-Crk and/or the activity of new downstream effectors in neuropeptide signal transduction (23). For example, c-Crk binds to a number of signaling proteins through its SH3 terminal domain, including C3G, a guanine nucleotide exchange factor for Rap-1, which is a small GTP-binding protein that induces mitogenesis in Swiss 3T3 cells (see Ref. 5). Thus the induction of complex formation between p130cas and c-Crk is a novel early event in neuropeptide-mediated signal transduction.

    SIGNAL TRANSDUCTION PATHWAYS LEADING TO P125FAK TYROSINE PHOSPHORYLATION: CYTOSKELETAL LINK

The molecular mechanisms by which GPCR activation leads to a rapid increase in p125fak tyrosine phosphorylation are only beginning to emerge. GI peptides that induce p125fak tyrosine phosphorylation also stimulate PKC activation and Ca2+ mobilization from internal stores (25). Although activation of PKC is a potential signaling pathway leading to enhanced tyrosine phosphorylation of p125fak, several lines of evidence indicate that neuropeptides stimulate tyrosine phosphorylation of p125fak through a signal transduction pathway that is largely independent of PKC (30). For example, downregulation of PKC by chronic treatment with phorbol esters or addition of a selective inhibitor of PKC completely blocks the increase in p125fak tyrosine phosphorylation induced by phorbol esters but does not impair this response to bombesin. Similarly, Ca2+ does not mediate neuropeptide-stimulated p125fak tyrosine phosphorylation (30). Consequently, the two major early signals generated by activation of phospholipase C are not responsible for neuropeptide stimulation of p125fak tyrosine phosphorylation in Swiss 3T3 cells. These findings distinguish the regulation of p125fak activity by GPCR agonists from that of the p125fak homologue Pyk2, which is a Ca2+-regulated tyrosine kinase in neural cells (19).

A salient feature of p125fak is its subcellular localization to focal adhesions that form at the terminals of actin stress fibers (3). Several recent reports (31) have implicated the Rho family, which belongs to the Ras-related small G protein superfamily, in the assembly of focal adhesion plaques and in the regulation of the actin cytoskeleton in Swiss 3T3 cells. Microinjection of Rho into these cells increases the formation of actin fibers and the assembly of focal adhesion plaques. Conversely, microinjection of botulinum C3 exoenzyme, an ADP ribosyltransferase that impairs Rho function, disrupts the actin filament network. GPCR agonists also promote a rapid increase in stress fibers and focal adhesions, an effect apparently mediated by Rho (25). Interestingly, bombesin-induced p125fak tyrosine phosphorylation is completely blocked by treatment with cytochalasin D, which selectively disrupts the actin flament network and prevents focal adhesion assembly (25, 30). In contrast, dissolution of microtubules did not exert any inhibitory effect on p125fak tyrosine phosphorylation. These findings suggest that p125fak tyrosine phosphorylation induced by bombesin is downstream to focal adhesion assembly and actin reorganization (Fig. 1).


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Fig. 1.   A putative pathway by which gastrointestinal (GI) peptide signals are transduced into tyrosine phosphorylation of focal adhesion proteins. See text for details. GI peptides induce many other early events emanating from activation of Galpha q, including phosphoinositide breakdown, protein kinase C activation, Ca2+ mobilization from internal stores, arachidonic acid release leading to prostaglandin synthesis, and serine/threonine phosphorylation of many cellular proteins. These events are not represented in this diagram, which focuses on the pathway leading to tyrosine phosphorylation of focal adhesion proteins. GPCR, G protein-coupled receptors; CNF, cytotoxic necrotizing factor; DNT, dermonecrotic toxin (these toxins activate Rho, promoting its GTP-bound state). C3, C3 exoenzyme from C. botulinum; ROK, Rho-associated coiled-coil-forming protein kinase; Cyt. D, cytochalasin D; MLCK, myosin light chain kinase; MLC-P, phosphorylated form of MLC.

Subsequent studies have indicated that treatment of Swiss 3T3 cells with C3 exoenzyme attenuates p125fak tyrosine phosphorylation induced by bombesin. Furthermore, addition of nonhydrolyzable GTP analogs to permeable Swiss 3T3 cells induces tyrosine phosphorylation of p125fak in a Rho-dependent manner (27). Additional experiments with bacterial toxins that selectively target and activate Rho, including cytotoxic necrotizing factor from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica, also indicate a Rho-dependent tyrosine phosphorylation of p125fak (see Ref. 18). These findings strongly suggest the existence of a pathway activated by GPCRs in which Rho is upstream of cytoskeletal reorganization and tyrosine phosphorylation of focal adhesion proteins, as depicted in Fig. 1.

Similar to the findings with p125fak, bombesin-induced tyrosine phosphorylation of paxillin and p130cas also occurs through a PKC- and Ca2+-independent pathway, which is critically dependent on the integrity of the actin cytoskeleton and focal adhesion plaques and requires functional Rho (5, 34). Therefore, tyrosine phosphorylation of paxillin, p130cas, and p125fak is coordinately regulated. It is possible that paxillin and p130cas are cellular substrates for p125fak (or for a p125fak/Src complex) in a neuropeptide-stimulated tyrosine kinase pathway (Fig. 1).

    CROSS TALK BETWEEN P125FAK AND ERK PATHWAY

The mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine kinases that relay mitogenic signals to the nucleus, thereby modulating the activity of transcription factors. The two best characterized isoforms, p42mapk (ERK-2) and p44mapk (ERK-1), are directly activated by phosphorylation on specific tyrosine and threonine residues by the dual-specificity ERK kinase (or MEK). GI peptides stimulate rapid ERK activation, typically via PKC or Ras-dependent pathways, depending on the cell type. For example, bombesin induces a rapid and striking stimulation of the ERKs in Swiss 3T3 cells through PKC (29). Because p125fak has been implicated in the pathway leading to ERK activation in integrin-stimulated cells, it is possible that p125fak tyrosine phosphorylation is involved in bombesin-mediated p42mapk/p44mapk activation. However, disruption of the cytoskeleton by cytochalasin D, at concentrations that completely prevent tyrosine phosphorylation of p125fak in response to bombesin, does not interfere with p42mapk/p44mapk activation by bombesin (28). Furthermore, bombesin also induces ERK activation in Swiss 3T3 cells placed in suspension, a condition that also impairs the increase in tyrosine phosphorylation in response to bombesin (24, 28). These results imply that ERK activation can be dissociated from p125fak tyrosine phosphorylation in bombesin-stimulated Swiss 3T3 cells. Reciprocally, recent evidence has implicated ERK activation in control of the actin cytoskeleton and cell migration via phosphorylation and activation of the myosin light chain (MLC) kinase (15) (Fig. 1). The elucidation of the contribution of the ERKs to cytoskeletal responses and tyrosine phosphorylation of focal adhesion proteins warrants further experimental work.

In contrast to p125fak, Pyk2 has been strongly implicated, in at least some cell types, in the pathway leading from GPCRs to ERK activation (7, 8). It appears that p125fak and Pyk2 play different roles in GPCR-mediated signal transduction.

    A MODEL OF RHO-DEPENDENT TYROSINE PHOSPHORYLATION OF FOCAL ADHESION PROTEINS

The molecular steps that link GPCRs for neuropeptides to Rho-dependent tyrosine phosphorylation of focal adhesion proteins remain undefined. It is clear that a single receptor subtype, e.g., the bombesin/GRP-preferring receptor, mediates coupling to PLC-beta as well as tyrosine phosphorylation of p125fak (6). An attractive model is that a single GPCR could couple to several G proteins, e.g., Gq, and thereby to PLC-beta , and to a separate trimeric G protein that could lead to activation of Rho and consequently to cytoskeletal responses and tyrosine phosphorylation of focal adhesion proteins. In this context, the demonstration that constitutive active mutants of Galpha 12 and Galpha 13 stimulate Rho-dependent biological responses, including the formation of actin stress fibers and focal adhesion assembly, is clearly relevant (11). This suggests that GPCRs couple to Rho via either Galpha 12 and/or Galpha 13. The activated forms of Galpha 12/Galpha 13 could activate or recruit Rho exchange factors that would promote the GTP-bound form of Rho in vivo and thereby lead to tyrosine phosphorylation of p125fak, p130cas, and paxillin via stress fiber formation and assembly of focal adhesions, as shown in Fig. 1. In line with this proposal, recent experimental work (20) has demonstrated that expression of constitutively active mutants of Galpha 12 and Galpha 13 can induce tyrosine phosphorylation of focal adhesion proteins via a Rho-dependent signal transduction pathway. These results support the hypothesis that GPCRs lead to tyrosine phosphorylation of p125fak, p130cas, and paxillin via Galpha 12/13 and Rho (Fig. 1).

An important step in the understanding of the mechanism(s) by which Rho promotes cytoskeletal responses has been the identification of the protein kinase ROK (that binds Rho-GTP), as a downstream target of Rho. Microinjection of a constitutively active form of ROK has been shown to induce the formation of actin stress fibers and focal adhesion plaques (1). Thus it is likely that ROK plays a physiological role in transducing Rho activation into cytoskeletal responses. The finding that ROK leads to MLC phosphorylation, either by inhibition of the 130-kDa myosin-binding subunit of myosin phosphatase that dephosphorylates MLC and/or by direct phosphorylation and activation of MLC (17), has suggested a molecular mechanism by which Rho-mediated ROK activation triggers cytoskeletal reorganization. It is known that MLC phosphorylation leads to myosin filament formation and stimulates interaction with actin (3). It has been proposed that the tension generated by the actin-myosin interaction plays a key role in promoting the formation of stress fibers and in the clustering of integrins to which they are attached, giving rise to observable focal adhesions in nonexcitable cells. The translocation of p125fak into nascent focal adhesions would induce its activation and autophosphorylation, as a result of clustering and/or conformational change (12). The inhibitory effects of cytochalasin D and botulinum C3 exoenzyme on the induction of p125fak, p130cas, and paxillin tyrosine phosphorylation induced by GPCR agonists fit nicely within this model (Fig. 1).

    NEUROPEPTIDES ACTIVATE SRC FAMILY TYROSINE KINASES INDEPENDENTLY FROM TYROSINE PHOSPHORYLATION OF P125FAK

The kinase activity of Src kinase family members (such as Src, Yes, and Fyn) is repressed when a key tyrosine residue in the COOH-terminal region (corresponding to Tyr527 of the chicken protein) is phosphorylated by Csk (reviewed in Ref. 23). Phosphorylation at Tyr527 creates a binding site for the Src SH2 domain and allows an intramolecular interaction that locks Src in an inactive conformation. Two mechanisms that may unlock and activate Src family members are currently considered. In one case, dephosphorylation of Tyr527 by a tyrosine phosphatase may destabilize the complex, releasing the SH2 domain and thereby activating the kinase activity. An alternative mechanism, involving competition for the SH2 domain of Src by a high-affinity allosteric ligand, would also lead to enzymatic activation of this kinase (23). In this context, autophosphorylation of p125fak at Tyr397 creates a putative competing binding site for the SH2 domain of Src and thus would lead to the formation of a signaling complex in which Src kinases are active. In fact, treatment of Swiss 3T3 cells with bombesin induced a rapid and transient increase in the kinase activity of the Src family of tyrosine kinases that was not dependent on either PKC or Ca2+ (24). Interestingly, cytochalasin D, at concentrations that profoundly inhibit p125fak tyrosine phosphorylation, does not impair the increase in Src family kinase activity induced by bombesin. Furthermore, bombesin also induces Src kinase family activation in Swiss 3T3 cells placed in suspension, a condition that also prevents the tyrosine phosphorylation of p125fak induced by bombesin (24). These findings indicate that Src family kinase activation can be dissociated from p125fak tyrosine phosphorylation in bombesin-treated Swiss 3T3 cells and demonstrate that two distinct signal transduction pathways lead to protein tyrosine phosphorylation in these cells (Fig. 1). Src activation is also induced by other GPCR agonists, and it has been implicated in promoting Ras-dependent ERK activation (7). As discussed above, Pyk2 initiates a distinct pathway involving tyrosine phosphorylation that is downstream of IP3-induced Ca2+ mobilization, at least, in certain cell types (7, 8, 19).

    BIOLOGICAL ROLE OF P125FAK, P130CAS, AND PAXILLIN TYROSINE PHOSPHORYLATION

The results revealing that regulatory peptides induce increases in the level of tyrosine phosphorylation of p125fak, paxillin, and p130cas suggest novel mechanisms of action of these agonists. These findings assume an added importance in view of increasing evidence implicating p125fak and p130cas in fundamental biological processes, including cell migration, proliferation, and neoplastic transformation. Gene disruption experiments have demonstrated a critical role of p125fak in embryonic development, cell migration, and turnover of focal adhesions (14). Furthermore, microinjection of a dominant-negative COOH-terminal fragment of p125fak, which displaces endogenous p125fak from focal adhesions and prevents its activation, inhibits cell motility and attenuates serum-induced stimulation of cellular DNA synthesis (10). A constitutively activated form of p125fak prevents apoptosis and induces neoplastic transformation in Madin-Darby canine kidney cells (9). In addition, there is increasing evidence linking overexpression of p125fak to the invasive properties of cancer cells.

The adaptor protein p130cas has also been implicated in agonist-stimulated mitogenesis (5) and in cell transformation and has recently been identified as a mediator of p125fak-mediated cell migration (4). Indeed, complex formation between p130cas and c-Crk has been proposed to act as a critical switch in stimulating cell migration (16). Interestingly, embryonic fibroblasts lacking Galpha 13 also display a greatly impaired migratory response to thrombin receptor activation (21), a GPCR that also promotes tyrosine phosphorylation of p125fak, p130cas, and paxillin (20). As discussed previously, Galpha 13 is one of the candidates that couple GPCRs to the tyrosine phosphorylation pathway via Rho activation. Collectively, these genetic findings are entirely consistent with the signal transduction pathway depicted in Fig. 1 and raise the attractive possibility that p125fak and p130cas are downstream targets of Galpha 13 in a signal transduction pathway that regulates cell migration in response to GPCR agonists during development, wound healing, and tumor metastasis.

In conclusion, the findings discussed here indicate that the novel pathway leading to tyrosine phosphorylation of focal adhesion proteins (Fig. 1) plays an important role in transducing GI peptide signals into migratory, proliferative, and antiapoptotic responses. An important task for the future will be to close the gaps in the pathway depicted in Fig. 1, verify the function of this pathway in cells from the GI tract, and define the cross talk and synergistic interactions between the serine/threonine and tyrosine phosphorylation cascades in the promotion of the multiple biological responses induced by GI peptides in their target cells.

    FOOTNOTES

*  Fifth in a series of invited articles on G Protein-Coupled Receptors in Gastrointestinal Physiology.

Address for reprint requests: E. Rozengurt, 900 Veteran Ave., Warren Hall Rm. 11-124, Dept. of Medicine, School of Medicine, Univ. of California, Los Angeles, CA 90095-1786.

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Am J Physiol Gastroint Liver Physiol 275(2):G177-G182
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society