Involvement of phosphorylation of tyr‐31 and tyr‐118 of paxillin in MM1 cancer cell migration

Abstract

We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA-ROCK pathway. It remains to be elucidated, however, how the signals from both LPA and FN are integrated into cell migration. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time-course immunoblot analysis with anti-phosphotyrosine antibodies (Abs). Consequently, tyrosine-phosphorylation of paxillin was obviously persistent after stimulation with FN ؉ LPA as compared to after stimulation with either alone. Cell migration plays an essential role in various physiological and pathological phenomena such as embryogenesis, wound repair, inflammation and invasion and metastasis of cancer cells. 1,2 Cell migration is primarily mediated by integrin binding to extracellular matrices (ECM) and regulated by organization of the actin cytoskeleton and the formation of focal adhesion complexes, which are induced through activation of the Rho-family small GTP-binding proteins. [3][4][5] Integrins not only act as a scaffold but also generate important signals leading to cell migration. Integrinmediated signaling events that regulate cytoskeletal organization require Rho-family GTPases and conversely integrin-mediated adhesion itself regulates Rho-family GTPases. 6 Cell motility is closely related to morphology, growth, 7 differentiation and survival, in which different signals from other cell surface receptors are also implicated. 8 It remains to be elucidated, however, how the integrin and growth-factor signaling pathways are integrated into cell migration.Paxillin (p68-kD) is one of the integrin assembly proteins and can interact directly with several integrin assembly proteins, including vinculin, talin, ␤1 integrin, focal adhesion kinase (FAK), c-Src and Csk. 9,10 Three human isoforms of paxillin (␣, ␤ and ␥) and 2 homologous murine isoforms (␣ and ␤) have been identified, 11,12 and the ␣ form (original paxillin) appears to play a more dominant role. 13 Integrin-mediated tyrosine phosphorylation also enables paxillin to interact with various signaling molecules: tyrosine residues (Y) 31 and 118 being especially predominant targets of phosphorylation by kinases and creating binding sites for the SH2 domain of adaptor protein Crk. 14 -16 Thus, paxillin plays a pivotal role in cell adhesion, migration and further oncogenic transformation. 9,10 Because several cytokines and growth factors also induce tyrosine phosphorylation of paxillin, 9,10 it is suggested that the signals from both cytokine-and growth-factor receptors and those from integrins converge on paxillin.In efforts to evaluate the invasive capacity of cancer cells and to understand the mechanisms of cancer-host interactions including transcellular (transmonolayer) migration of cancer cells, we have developed an in vitro system: rat ascites hepatoma MM1 cells are allowed to invade a mesothelial cel...

Paxillin: Adapting to Change

“…Importantly, Arf1-dependent paxillin shuttling between the Golgi and focal adhesions has been described (191), with the ArfGAPs, GIT2short, and ASAP2/PAP␣ implicated in this process (128,175). Other critical mediators of Golgi reorientation/polarization are Src (120) and p120RasGAP (135), which are paxillin binding partners (112,302). p120RasGAP is essential for polarization likely through the temporal-spatial regulation of Rho activities (135).…”

“…p120RasGAP is essential for polarization likely through the temporal-spatial regulation of Rho activities (135). Paxillin, via pY31/118-p120RasGAP binding and resulting dissociation of p190RhoGAP from p120RasGAP, has been proposed as a mechanism to regulate p190RhoGAP activity (112). Through this exchange paxillin may contribute both to the inhibition of Rho function at the leading edge to promote lamellipodial extension and activation of Cdc42 in the context of cell polarity.…”

Ipriflavone inhibits osteolytic bone metastasis of human breast cancer cells in a nude mouse model

“…Cells were then incubated with 100 ng/ml recombinant human EGF (Austral Biologicals, San Ramon, CA) for 5 min, washed with cold PBS and solubilized with lysis buffer. 35 Protein (15 g) from each extract was separated by 7.5% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with anti-EGFR antibody (1:1,000 dilution; Cell Signaling Technology, Beverly, MA) or phosphorylation site-specific anti-phospho-EGFR (Tyr 845 ) antibody (1:1,000 dilution, Cell Signaling Technology) in TTBS at 4°C overnight.…”

Expression of paxillin and FAK mRNA and the related clinical significance in esophageal carcinoma

“…Paxillin is associated with integrin and its related cellular and extracellular matrix molecules. As such, paxillin helps to regulate cell migration, dissemination and other functions, and enhances tumor cell invasion and metastasis (1,2). Focal adhesion kinase (FAK) is distributed in the cellular focal adhesion sites and plays a crucial role in the regulation of tumor cell migration (3,4).…”