Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine.
Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.
Peters
J D
JD
Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Furlong
M T
MT
Asai
D J
DJ
Harrison
M L
ML
Geahlen
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007075:Immunoglobulin M / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000072377:Syk Kinase; D014404:Tubulin / Q000378:metabolism*
D004792:Enzyme Precursors; D007075:Immunoglobulin M; D047908:Intracellular Signaling Peptides and Proteins; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D019000:Phosphotyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse