Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Src is activated in response to a variety of growth factors and hormones that bind G protein-coupled receptors (GPCRs), and its activity is regulated by phosphorylation at key sites, including the autophosphorylation site Tyr-418 and the inhibitory site Tyr-529. To better understand the mechanisms controlling Src activation, we examined Src phosphorylation in Swiss 3T3 fibroblasts stimulated with bombesin and in IEC-18 intestinal epithelial cells stimulated with angiotensin II (Ang II). Phosphorylation at Src Tyr-418, the activation loop site, was rapidly and markedly increased after GPCR agonist addition in both cell types. However, treatment of intact cells with the selective Src family kinase inhibitor PP2, at concentrations which abolished Src-mediated phosphorylation of focal adhesion kinase (FAK) at Tyr-577, unexpectedly led to increased phosphorylation at Src Tyr-418 and diminished phosphorylation at Tyr-529. In Swiss 3T3 cells, PP2 enhanced Tyr-418 phosphorylation after 1 min of bombesin stimulation, while in IEC-18 cells, PP2 increased Ang II-stimulated Tyr-418 phosphorylation at all times tested. These results imply that a distinct, non-Src family kinase may be responsible for phosphorylating Src at Tyr-418 in intact fibroblasts and epithelial cells stimulated by GPCR agonists.
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PMID:Bombesin and angiotensin II rapidly stimulate Src phosphorylation at Tyr-418 in fibroblasts and intestinal epithelial cells through a PP2-insensitive pathway. 1545 Oct 29

Although amidated forms of gastrin-releasing peptide (GRP) have been identified as autocrine growth factors in small cell lung cancer, their role in the development and progression of colorectal carcinoma is less clear. In addition, the biological activity of non-amidated gastrin-releasing peptide has not been investigated in colorectal carcinoma cells. We therefore investigated the effect of bombesin (a homologue of gastrin-releasing peptide) on proliferation, migration and inositol phosphate production in the human colorectal carcinoma cell line DLD-1, and determined the ability of gastrin-releasing peptide receptor antagonists to inhibit these effects. We also compared the biological activities of amidated and non-amidated GRP in the same assays. Treatment with either bombesin, or amidated or non-amidated GRP resulted in significant increase in proliferation, and in migration in a wound-healing assay. Both the mitogenic and migratory effects of amidated and non-amidated forms were inhibited by the GRP receptor antagonist [D-Phe(6), Leu-NHet(13), des-Met(14)]-bombesin(6-13). The presence of GRP receptor mRNA and GRP binding sites in three colorectal carcinoma cell lines was demonstrated by RT-PCR and by binding of radiolabelled bombesin, respectively. Transfection of DLD-1 cells with a dominant negative phosphatidylinositol 3-kinase did not affect bombesin-stimulated cell proliferation, but inhibited bombesin-stimulated cell migration. Bombesin and GRPgly activated phospholipase C, mitogen-activated protein kinase and focal adhesion kinase. We conclude that both amidated and non-amidated forms of gastrin-releasing peptide accelerate proliferation and migration of DLD-1 human colorectal carcinoma cells via the gastrin-releasing peptide receptor, but that phosphatidylinositol 3-kinase is only involved in the cell migration signalling pathway. Our results suggest a potential role for gastrin-releasing peptide receptor antagonists in the management of colorectal carcinoma.
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PMID:Stimulation of proliferation and migration of a colorectal cancer cell line by amidated and glycine-extended gastrin-releasing peptide via the same receptor. 1549 3

Neuroendocrine (NE) cells are found in prostate tumors, and their incidence is considered a promising prognostic indicator for the development of androgen-independent disease. NE cells are derived from non-NE prostate cancer cells and secrete factors that can act in a paracrine manner to stimulate the survival, growth, motility, and metastatic potential of prostatic carcinoma cells. Factors such as IL-6, epinephrine, and forskolin induce NE differentiation in prostate cancer cells; the mechanisms involve increases in intracellular cAMP, protein kinase A (PKA) activation and reduced intracellular calcium levels. Transcription factors implicated in the acquisition of NE characteristics by prostate cancer cells include STAT3, CREB, EGR1, c-fos, and NF-kappaB. Expression of Chromogranin A, neuron-specific enolase, bcl-2, and the androgen receptor are modulated during NE differentiation and serve as molecular markers for NE cells. Most importantly, NE cells secrete neuropeptides, such as bombesin, neurotensin, PTHrP, serotonin, and calcitonin, which trigger growth and survival responses in androgen-independent prostate cancer cells. Prostate cancer cell receptors that play a role in these processes include the gastrin-releasing peptide (GRP) receptor, neurotensin receptors, and the epidermal growth-factor receptor (EGFR). Signal-transduction molecules activated by these neuropeptides include Src, focal adhesion kinase (FAK), ERK, and PI3K/Akt, with subsequent activation of Elk-1, NF-kappaB, and c-myc transcription factors. A multitude of genes are then expressed by prostate cancer cells, which are involved in proliferation, anti-apoptosis, migration, metastasis, and angiogenesis. Targeting of these pathways at multiple levels can be exploited to inhibit the process by which NE cells contribute to the progression of androgen-independent, treatment-refractory prostate cancer.
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PMID:Neuroendocrine cells in prostate cancer. 1566 58

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with the G protein-coupled receptor agonists bombesin, vasopressin, or bradykinin induced an extremely rapid (within 5 s) increase in FAK phosphorylation at Ser-843. The phosphorylation of this residue preceded FAK phosphorylation at Tyr-397, the major autophosphorylation site, and FAK phosphorylation at Ser-910. Treatment of intact cells with ionomycin stimulated a rapid increase in FAK phosphorylation at Ser-843, indicating that an increase in intracellular Ca2+ concentration ([Ca2+]i) is a potential pathway leading to FAK-Ser-843 phosphorylation. Indeed, treatment with agents that prevent an agonist-induced increase in [Ca2+]i (e.g. thapsigargin or BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), interfere with calmodulin function (e.g. trifluoperazine, W13, and W7), or block Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation (KN93) or expression (small interfering RNA) abrogated the rapid FAK phosphorylation at Ser-843 induced by bombesin, bradykinin, or vasopressin. Furthermore, activated CaMKII directly phosphorylated the recombinant COOH-terminal region of FAK at a residue equivalent to Ser-843. Thus, our results demonstrate that G protein-coupled receptor activation induces rapid FAK phosphorylation at Ser-843 through Ca2+, calmodulin, and CaMKII.
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PMID:G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at Ser-843. Mediation by Ca2+, calmodulin, and Ca2+/calmodulin-dependent kinase II. 1584 48

Clinical evidence links neuroendocrine differentiation (NED) to prostate cancer progression. In the prostate carcinoma PC-3 cell model, the action of the gastrin releasing peptide (GRP) analog, bombesin (BN), on the activation of focal adhesion kinase (FAK) and invasiveness suggests that this kinase might favor metastasis. Given that components of the FAK signalling pathway are also up regulated in prostate cancer, the aim of the present investigation was to test if FAK function is required for BN-induced motility in PC-3 cells. In wound assays designed to investigate the fate of FAK in cells undergoing BN-induced motility, it was observed that BN treatment resulted in relocalization of FAK in focal contacts concomitantly with its tyrosine phosphorylation on residue 397 (FAK [pY(397)]) and with the formation of actin lamellipodia. Moreover, BN-induced cell motility was significantly reduced in the presence of FAK inhibitors (either anti-FAK [pY(397)] antibody or FRNK, the FAK-related non-kinase). Altogether, these observations point towards a critical role for FAK in the action of BN on PC-3 cell motility.
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PMID:Focal adhesion kinase is required for bombesin-induced prostate cancer cell motility. 1586 27

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr(397), Tyr(407), Tyr(861), and Tyr(925) but not Tyr(576) or Tyr(577). To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at approximately 2 h, and maximal levels were observed at approximately 6 h postwounding. The GRP-specific antagonist [d-Phe(6)]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.
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PMID:Transient upregulation of GRP and its receptor critically regulate colon cancer cell motility during remodeling. 1589 Jul 13

Gastrointestinal peptides including mammalian bombesin-like peptides, cholecystokinin (CCK), gastrin, and neurotensin stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a number of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation. These agonists bind to G protein-coupled receptors (GPCRs) that promote Galpha q-mediated activation of beta isoforms of phospholipase C to produce two second messengers: Inositol (1,4,5) trisphosphate {Ins (1, 4, 5) P3} that mobilises Ca2+ from internal stores, and diacylglycerol that activates the classic and new isoforms of the protein kinase C (PKC) family. PKCs play a critical part in transducing bombesin/gastrin releasing peptide (GRP) receptor signals into activation of protein kinase cascades. Protein kinase D (PKD), a serine/threonine protein kinase with distinct structural and enzymological properties, is activated by phosphorylation in living cells through a new PKC-dependent signal transduction pathway. GPCR agonists including bombesin/GRP induce a rapid and striking activation of PKD by PKC. These results indicate that PKD functions downstream from PKCs and identify a new phosphorylation cascade that is activated by gastrointestinal peptide agonists. The bombesin/GRP GPCR also promotes rapid Rho-dependent assembly of focal adhesions, formation of actin stress fibres and tyrosine phosphorylation of multiple cellular proteins. We identified p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (CAS) and paxillin as prominent targets of gastrointestinal peptide-stimulated tyrosine phosphorylation and developed a model that envisages a G12/Rho-dependent pathway connecting GPCR activation to the tyrosine phosphorylation of these focal adhesion proteins. Separate pathways mediate gastrointestinal peptide stimulation of additional tyrosine kinase pathways including transactivation of Src and epidermal growth factor receptor (EGFR). Tyrosine phosphorylation has a critical role in gastrointestinal peptide-induced cellular migration and cooperates with Gq-stimulated events to promote mitogenesis. The growth-promoting effects of neuropeptides and the elucidation of the signalling pathways that mediate their effects assume an added importance because these agonists and their receptors are increasingly implicated in sustaining the proliferation of clinically aggressive solid tumours including those from lung, pancreas, and colon.
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PMID:Gastrointestinal peptide signalling in health and disease. 1614 98

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.
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PMID:Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism. 1636 65

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.
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PMID:Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase. 1665 49

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.
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PMID:Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth. 1741 80


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