Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive.
Tyrosine phosphorylation of paxillin regulates actin cytoskeleton-dependent changes in cell morphology and motility in adherent cells. In this report we investigated the involvement of paxillin tyrosine phosphorylation in the regulation of actin cytoskeleton-dependent polarization and motility of a non-adherent IL-3-dependent murine pre-B lymphocytic cell line Baf3. We also assessed the effect of phorbol myristate acetate (PMA), a phorbol ester analogous to those currently in clinical trials for the treatment of leukemia, on paxillin phosphorylation. Using tyrosine-to-phenylalanine phosphorylation mutants of paxillin and phosphospecific antibody we demonstrated that IL-3 stimulated phosphorylation of paxillin tyrosine residues 31 and 118, whereas the tyrosines 40 and 181 were constitutively phosphorylated. Phosphorylation of paxillin residues 31 and 118 was required for cell polarization and motility. In the presence of IL-3, PMA dramatically reduced the phosphorylation of residues 31 and 118, which was accompanied by inhibition of cell polarization and motility. This PMA effect was partially recapitulated by expression of exogenous tyrosine 31 and 118 mutants of paxillin. We also demonstrated that PMA inhibited the IL-3-induced and activation-dependent tyrosine phosphorylation of focal adhesion kinase. Thus, our results indicate that phosphorylation of paxillin tyrosine residues 31 and 118 regulates actin-dependent polarization and motility of pre-B Baf3 cells, both of which could be inhibited by PMA. They also suggest that inhibition of upstream signaling by PMA contributes to the decrease of paxillin phosphorylation and subsequent changes in cell morphology.
Romanova
Larisa Y
LY
Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA. iromanova@partners.org
Hashimoto
Shigeru
S
Chay
Kee-Oh
KO
Blagosklonny
Mikhail V
MV
Sabe
Hisataka
H
Mushinski
J Frederic
JF
D000199:Actins / Q000378:metabolism; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007377:Interleukin-3 / Q000378:metabolism; D008214:Lymphocytes; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014443:Tyrosine / Q000737:chemistry*
D000199:Actins; D003598:Cytoskeletal Proteins; D007377:Interleukin-3; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D014443:Tyrosine; D010744:Phosphoric Monoester Hydrolases; D013755:Tetradecanoylphorbol Acetate