Chicken and Mouse Focal Adhesion Kinases Are Similar in Structure at Their Amino Termini

Devor, B. B.; Zhang, Xiaoe; Patel, Sheetal; Polte, Thomas R.; Hanks, Steven K.

doi:10.1006/bbrc.1993.1160

Cited by 16 publications

(5 citation statements)

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“…Anti-Pyk2 antibodies were then affinity purified from the antiserum using GSTϪPk2. C immobilized on glutathione-Sepharose as an affinity matrix, as described previously [24]. The anti-Pyk2 antibodies did not crossreact with FAK in a variety of cell types including the RASM cells (Zheng, C., unpublished results).…”

Section: Methodsmentioning

confidence: 96%

Differential stimulation of proline‐rich tyrosine kinase 2 and mitogen‐activated protein kinase by sphingosine 1‐phosphate

Guo

Zheng

Martín‐Padura

et al. 1998

European Journal of Biochemistry

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Sphingosine 1-phosphate (SphP), a metabolite of cellular sphingolipids, has been shown to induce cell proliferation by activating the mitogen-activated protein kinase (MAPK) pathway. Proline-rich tyrosine kinase 2 (Pyk2) is a novel cytosolic tyrosine kinase which mediates activation of the MAPK or c-Jun N-terminal kinase (JNK) signaling pathways in response to a variety of stimuli that elevate intracellular calcium. In this report, we show that SphP stimulates both tyrosine phosphorylation of Pyk2 and MAPK activation in a transient and dose-dependent manner in rat aortic smooth muscle cells. Further studies indicate that Pyk2 phosphorylation, but not MAPK activation, is dependent on a pertussis toxinsensitive G-protein-coupled receptor as well as partially on actin cytoskeleton. In addition, both intracellular calcium mobilization and protein kinase C (PKC) are required for optimal Pyk2 phosphorylation while either calcium increase or PKC activation is sufficient for MAPK activation in response to SphP. Finally, we show that a tyrosine kinase(s) other than Pyk2 is necessary for MAPK activation by SphP. Together, these results suggest that SphP stimulates tyrosine phosphorylation of Pyk2 through a G-protein coupled receptor, which is dissociated from its activation of the MAPK pathway in these cells.

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Section: Methodsmentioning

confidence: 96%

Differential stimulation of proline‐rich tyrosine kinase 2 and mitogen‐activated protein kinase by sphingosine 1‐phosphate

Guo

Zheng

Martín‐Padura

et al. 1998

European Journal of Biochemistry

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“…Plasmids pCMV-Myc-FAK and pCMV-Myc-FRNK were constructed by PCR amplification utilizing chicken fak or frnk cDNA (14,33,34) as a template, and the resulting DNA fragments were individually inserted into pCMV(Myc) followed by DNA sequence verification. The construction of eps8 siRNA expression construct, pS-hEps8 (5Ј-GAT CCC GCT GTG ATG CAT TCA TGC ATT CAA GAG ATG CAT GAA TGC ATC ACA GCT TTT TTG GAA A-3Ј), was similarly carried out as described previously (7) according to human eps8 cDNA sequence (GenBank TM accession number: U12535).…”

Section: Methodsmentioning

confidence: 99%

EPS8 Facilitates Cellular Growth and Motility of Colon Cancer Cells by Increasing the Expression and Activity of Focal Adhesion Kinase

Maa

Lee

Chen

et al. 2007

Journal of Biological Chemistry

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In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.The signal transduction of the epidermal growth factor receptor (EGFR) 2 is important for normal cell physiology (1, 2). During the search for novel EGFR substrates, Eps8 (EGFR pathway substrate number 8) as suggested by its designation was originally identified as a putative EGFR target devoid of phosphotyrosine-binding SH2 domain (3). Among its 97-and 68-kDa isoforms, only the former is well characterized and thus referred to as Eps8. Later, Eps8 also is the substrate for Src tyrosine kinase (4). In addition to tyrosyl phosphorylation, expression of Eps8 is also affected by Src activity (4, 5). Remarkably, its aberrant overexpression in murine fibroblasts can lead to cellular transformation (6), and of note, Eps8 overexpression contributes to Src-mediated transformation (7).As an adaptor protein, Eps8 contains several structural features such as a split pleckstrin homology, a putative nuclear targeting sequence, a central SH3 domain, and several prolinerich regions. Although the split pleckstrin homology confers the ability of Eps8 to associate with plasma membrane in response to serum stimulation and conveys signals to ERK activation (6), Eps8 can also complex with Abi-1/E3b1 and RN-tre separately through its SH3 domain (8, 9). By interacting with Abi-1 or RN-tre, Eps8 integrates signals leading to actin cytoskeleton via Rac and receptor endocytosis via Rab5, respectively (10, 11). Recently, IRSp53 has been demonstrated as an Eps8-binding protein whose complex with Eps8 reinforces Rac activation and cell migration in fibrosarcoma cells (12). Besides, Eps8 is also identified as an actin capper, which is capable of regulating actin-based motility (13).Focal adhesion kinase (FAK) is an intracellular tyrosine kinase localized prominently within focal adhesion (14, 15) and participates in a variety of integrin-elicited biological ac...

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“…Anti-Pyk2 antibodies were then affinity-purified from the antiserum using GST-Pyk2.C immobilized on glutathione-Sepharose as an affinity matrix, as described previously (10). Rabbit polyclonal anti-FAK serum and mouse monoclonal antibody 12CA5 against an epitope of the hemagglutinin (HA) protein were as described (11,12).…”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of Pyk2 and Focal Adhesion Kinase (FAK)

Zheng¹,

Xing

Bian

et al. 1998

Journal of Biological Chemistry

129

102

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Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.Proline-rich tyrosine kinase 2 (Pyk2 1 ; also known as CAK␤, RAFTK, and CADTK) is a recently described cytoplasmic tyrosine kinase that is related to the focal adhesion kinase (FAK) (1-4). Recent studies have shown that Pyk2 is involved in calcium-induced regulation of ion channel and mitogen-activated protein kinase activation (1), stress-induced c-Jun Nterminal kinase activation (5), and Src-mediated activation of the mitogen-activated protein kinase signaling pathway in PC12 cells (6). Although these studies indicate that Pyk2 is involved in several signal transduction pathways, many questions concerning the regulation and function of Pyk2, especially the role of Pyk2 in cell adhesion, remain unanswered.Pyk2 and FAK share a similar structural organization with a tyrosine kinase domain flanked by non-catalytic domains at both the N and C termini. These two kinases are approximately 60% identical in the central catalytic domain and share approximately 40% identity in both the N-and C-terminal domains (1-3). Because of the high sequence homology and similar overall organization between Pyk2 and FAK, it is especially interesting to compare the regulation of Pyk2 with that of FAK, in particular their responses to integrin-mediated cell adhesion. Several recent reports have presented somewhat conflicting data regarding regulation of Pyk2 by integrin-mediated cell adhesion in different cell types. It has been reported that Pyk2 displays an integrin-dependent phosphorylation and is localized in focal contacts in B lymphocytes, megakaryocytes, and transfected COS cells (7,8); however, in transfected 3Y1 cells, Pyk2 phosphorylation is not increased upon plating on fibronectin (FN) an...

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Chicken and Mouse Focal Adhesion Kinases Are Similar in Structure at Their Amino Termini

Cited by 16 publications

References 0 publications

Differential stimulation of proline‐rich tyrosine kinase 2 and mitogen‐activated protein kinase by sphingosine 1‐phosphate

Differential stimulation of proline‐rich tyrosine kinase 2 and mitogen‐activated protein kinase by sphingosine 1‐phosphate

EPS8 Facilitates Cellular Growth and Motility of Colon Cancer Cells by Increasing the Expression and Activity of Focal Adhesion Kinase

Differential Regulation of Pyk2 and Focal Adhesion Kinase (FAK)

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