Phosphorylation of paxillin at threonine 538 by PKCdelta regulates LFA1-mediated adhesion of lymphoid cells.
We investigated the PKCdelta-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCdelta directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.
Romanova
Larisa Y
LY
Laboratory of Cancer Biology and Genetics, Cell and Cancer Biology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. romanovl@dc37a.nci.nih.gov
Holmes
Gibran
G
Bahte
Svenja K
SK
Kovalchuk
Alexander L
AL
Nelson
Patrick J
PJ
Ward
Yvona
Y
Gueler
Faikah
F
Mushinski
J Frederic
JF
D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007377:Interleukin-3 / Q000494:pharmacology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D008214:Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D000199:Actins; D007377:Interleukin-3; D016169:Lymphocyte Function-Associated Antigen-1; D051419:Paxillin; D010769:Phosphothreonine; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate
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