Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells.
Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.
Fernandez
J A
JA
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA.
Keshvara
L M
LM
Peters
J D
JD
Furlong
M T
MT
Harrison
M L
ML
Geahlen
R L
RL
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis / Q000276:immunology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D000072377:Syk Kinase; D014404:Tubulin / Q000378:metabolism*
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins; D010750:Phosphoproteins; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic; D014404:Tubulin; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase