Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

doi:10.1016/j.cellsig.2006.11.004

Cellular Signalling

Volume 19, Issue 5, May 2007, Pages 1000-1010

https://doi.org/10.1016/j.cellsig.2006.11.004 Get rights and content

Abstract

A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.

Introduction

A large body of evidence has demonstrated that a rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase p125 focal adhesion kinase (FAK) is a prominent early event in fibroblasts stimulated by diverse signaling molecules that regulate cell proliferation, migration and survival, including mitogenic agonists that act via G protein-coupled receptors (GPCRs), growth factors, integrin clustering induced by cell adhesion, bacterial toxins, and activated variants of pp60^src [1], [2], [3], [4], [5], [6]. Autophosphorylation of FAK at Tyr-397 [7], [8], located N-terminal to the catalytic domain, creates a binding site for the tyrosine kinase Src and other downstream signaling effectors, including PI 3-kinase and phospholipase Cγ [6]. Subsequent Src-mediated phosphorylation of FAK at Tyr-576, Tyr-577, Tyr-861 and Tyr-925, is important for the maximal activation of FAK and phosphorylation at Tyr-397 [4], [9], [10]. This model of FAK activation and Tyr-397 phosphorylation is thought to be of central importance for FAK-dependent signaling. The biological importance of FAK-mediated signal transduction is underscored by the fact that this tyrosine kinase plays a fundamental role in embryonic development [11], [12] and in the control of cell migration [9], [12], [13], [14], [15], [16], [17], [18], [19], cell cycle progression [20] and apoptosis [21], [22], [23], [24]. Furthermore, there is increasing evidence linking overexpression of FAK to the invasive properties of cancer cells [25], [26], [27], [28], [29], [30], [31].

More recently, it has become clear that FAK is also phosphorylated at multiple serine residues [32], [33], [34], [35], [36], [37]. The proximity of these phosphorylated serine residues to sites at which FAK interacts with other proteins suggests a possible function in the regulation of the assembly of FAK signaling complexes [6]. Recent results from our laboratory demonstrated that FAK phosphorylation at Ser-910 is strikingly stimulated by GPCR agonists, tumor promoting phorbol esters and growth factors through an ERK-dependent pathway in Swiss 3T3 fibroblasts [34], [35], [36]. In another study, we demonstrated that stimulation of 3T3 fibroblasts with bombesin, bradykinin or vasopressin induced a rapid and transient increase in FAK phosphorylation at Ser-843 through a Ca²⁺/calmodulin/CaMKII-dependent pathway [37]. These findings support the hypothesis that FAK phosphorylation at both, tyrosine and serine residues is modulated by a variety of stimuli but do not exclude the possibility that FAK phosphorylation at tyrosine and serine residues could be regulated differentially in some cell types.

FAK is expressed in intestinal epithelial cells and re-plating suspended cultures of these cells onto extracellular matrix proteins induces tyrosine phosphorylation of this enzyme and its association with focal adhesions [38], [39]. However, many groups noticed that the basal level of tyrosine phosphorylated FAK is high in epithelial cells attached to a substratum, eg T84 cells [40], IEC-18 cells [41], SW 620 [42] and liver epithelial cells [43]. These results imply that FAK tyrosine phosphorylation is constitutively active in attached epithelial cells and thus, it might not be further enhanced in response to receptor activation by external soluble stimuli. These considerations prompted us to consider that stimulation of epithelial cells with GPCR agonists or ligands of tyrosine kinase receptors could increase FAK phosphorylation differentially at serine rather than at tyrosine residues.

The results presented here show that stimulation of undifferentiated intestinal epithelial IEC-18 cells with multiple agonists, including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol-12,13-dibutyrate (PDB) or EGF induced a striking stimulation of FAK phosphorylation at Ser-910 via an ERK-dependent pathway. In contrast, none of these stimuli promoted a significant increase of FAK phosphorylation at Tyr-397 in these cells. These results were extended using polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but only slight stimulation of FAK at Tyr-397 in these cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that FAK phosphorylation at Ser-910 and Ser-843, rather than at Tyr-397, is strikingly increased in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in rat and human intestinal epithelial cells.

Section snippets

Cell culture

Both IEC-18 cells and T84 cells were purchased from American Type Culture Collection. Stock cultures of IEC-18 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO₂ and 90% air at 37 °C. For experimental purposes, cells were plated into 35-mm dishes at 1 × 10⁵ cells per dish, in DMEM containing 5% FBS and were allowed to grow to confluency (6–8 days) before use.

The colonic epithelial cell line

ANGII induces FAK phosphorylation at Ser-910 in IEC-18 cells

ANGII binds to specific endogenous receptors in intestinal epithelial IEC-18 cells [44] and induces multiple signaling pathways and biological responses in these cells, including DNA synthesis and proliferation [45], [46], [47], [48], [49]. As shown by the results presented in Fig. 1A (control), stimulation with ANGII also markedly increased the migration of IEC-18 cells into the denuded area of a razor blade wound. In order to determine whether FAK is necessary for the migration of IEC-18

Discussion

Numerous studies have demonstrated that a rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in cells stimulated by multiple signaling molecules that regulates cell proliferation, migration, and apoptosis [3], [6], [52]. On the contrary, much less is known about the regulation of FAK phosphorylation at serine residues by external stimuli including GPCR agonists and growth factors. Interestingly, both Ser-910 and Ser-843, located in

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^☆: This work was supported by NIH Grant DK 56930, DK 55003, NCI Grant P50 CA90388 and P30 DK41301, Xiaohua Jiang was supported by NCI Grant CA09056.

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Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells☆

Abstract

Introduction

Section snippets

Cell culture

ANGII induces FAK phosphorylation at Ser-910 in IEC-18 cells

Discussion

Cell

Prog. Biophys. Mol. Biol.

Biochim. Biophys. Acta

J. Biol. Chem.

Mol. Cells

J. Biol. Chem.

Am. J. Pathol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signal.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Curr. Opin. Cell Biol.

Cell

Cancer Surv.

Bioessays

J. Cell Sci.

Mol. Cell. Biol.

Mol. Cell. Biol.

Mol. Cell. Biol.

Nature

J. Cell Sci.

Mol. Cell. Biol.

J. Cell Sci.

EMBO J.