Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.     

Involvement of G proteins of the Rho family in the regulation of Bcl-2-like protein expression and caspase 3 activation by Gastrins

Gastrins, including amidated gastrin (Gamide) and glycine-extended gastrin (Ggly), are known to accelerate the growth of gastric and colorectal cancer cells by stimulation of proliferation and inhibition of apoptosis. Gamide controls apoptosis by regulation of proteins of the Bcl-2 family and by regulation of the activation of caspases. However the interactions between Ggly and proteins of the Bcl-2 family and caspases are not known. Since in other systems G proteins of the Rho family inhibit apoptosis via interaction with proteins of the Bcl-2 family, leading to changes in caspase activities, we have compared the role of Rho family G proteins in regulation of Bcl-2-like (Bad/Bax/Bcl-xl) protein expression and caspase 3 activation by Ggly and Gamide. The effects of the specific inhibitors C3 (for Rho) and Y-27632 (for ROCK), and of dominant negative mutants of Rac, Cdc42 and PAK, were investigated in the gastric epithelial cell line IMGE-5. Apoptosis was induced by serum starvation and confirmed by annexin V staining and caspase 3 activation. Ggly inhibits caspase 3 activation via a Bcl-2-like protein-mediated pathway which requires activation of both Rho/ROCK and Rac/Cdc42/PAK. Gamide inhibits caspase 3 activation via redundant Bcl-2-like protein-mediated pathways which involve alternative activation of Rac/Cdc42/PAK and Rho/ROCK. Gamide and Ggly differentially activate members of Rho family G proteins which in turn regulate different proteins of the Bcl-2 family leading to changes in caspase 3 activity. The findings offer potential targets for blocking the growth-stimulating effects of these gastrins.     

The C-terminal flanking peptide of progastrin induces gastric cell apoptosis and stimulates colonic cell division in vivo

Progastrin (PG) is processed into a number of smaller peptides including amidated gastrin (Gamide), non-amidated glycine-extended gastrin (Ggly) and the C-terminal flanking peptide (CTFP). Several groups have reported that PG, Gamide and Ggly are biologically active in vitro and in vivo, and are involved in the development of gastrointestinal cancers. CTFP is bioactive in vitro but little is known of its effects in vivo. This study investigated the bioactivity of CTFP in vivo in normal tissues using gastrin deficient (GASKO) mice and in two mouse models of cancer (SCID mice bearing xenograft tumors expressing normal or knocked-down levels of gastrin and a mouse model of hepatic metastasis). As with Ggly, CTFP treatment stimulated colonic proliferation in GASKO mice compared to control. CTFP also significantly increased apoptosis in the gastric mucosa of male GASKO mice. CTFP did not appear to effect xenograft growth or the incidence of liver metastases. This is the first demonstration that CTFP has specific biological activity in vivo in the colon and stomach.     

Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.     

The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.     

Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase's activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.     

YES,a Src Family Kinase,Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

Direct interaction of p21-activated kinase 4 with PDZ-RhoGEF, a G protein-linked Rho guanine exchange factor

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.     

Cadherin-mediated cellular signaling

Recent cadherin studies focusing on cellular signaling have shown that several pathways are activated by cadherin-mediated cell-cell contact. Cadherin-mediated contacts activate Rho family GTPases, regulate the availability of beta-catenin to participate in Wnt signaling, and function in receptor tyrosine kinase signaling. Although different classical cadherins bind to the same cytosolic proteins via their cytoplasmic tails, one message that is clear from the recent literature is that downstream signals emanating from cadherin-mediated contacts are both cadherin-specific and cell-context-specific.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.     

Peroxisome proliferator-activated receptor gamma promotes epithelial to mesenchymal transformation by Rho GTPase-dependent activation of ERK1/2

Peroxisome proliferator-activated receptor gamma (PPARgamma) causes epithelial to mesenchymal transformation (EMT) in intestinal epithelial cells, as evidenced by reorganization of the actin cytoskeleton, acquisition of a polarized, mesenchymal cellular morphology, increased cellular motility, and colony scattering. This response is due to activation of Cdc42, resulting in p21-activated kinase-dependent phosphorylation and activation of MEK1 Ser(298) and activation of ERK1/2. Dominant negative MEK1, MEK2, and ERK2 block PPARgamma-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by PPARgamma. PPARgamma also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPARgamma induces the p110alpha subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPARgamma-dependent phosphorylation of MEK1 Ser(298), activation of ERK1/2, and EMT. We conclude that PPARgamma regulates the motility of intestinal epithelial cells through a mitogen-activated protein kinase cascade that involves PI3K, Cdc42, p21-activated kinase, MEK1, and ERK1/2. Regulation of cellular motility through Rho family GTPases has not been previously reported for nuclear receptors, and elucidation of the mechanism that accounts for the role of PPARgamma in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve

To clarify the presence of the Rho family of small GTPases p21-activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co-immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co-immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42-pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.