CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase.
The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45(+) and CD45(-) T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.
Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada.
Nelson K Y
NK
Wong
Jacqueline C Y
JC
Lai
Nina
N
Maeshima
Pauline
P
Johnson
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D018960:Hyaluronan Receptors / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008870:Microtubules; D017239:Paclitaxel / Q000494:pharmacology; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology / Q000502:physiology; D050257:Tubulin Modulators / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D000199:Actins; D018960:Hyaluronan Receptors; D050257:Tubulin Modulators; D058539:Phosphatidylinositol 3-Kinase; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens; D010738:Type C Phospholipases; D017239:Paclitaxel; D002118:Calcium