We have demonstrated that a 72-kDa non-receptor-type protein-tyrosine kinase (~7 2 "~" ) was coimmunoprecipitated with membrane IgM in digitonin lysates of porcine tonsillar cells and was rapidly activated following the engagement of membrane IgM. This activation was occurred within 5 s, even in the presence of EGTA and 5,5'-dimethyl-bis-(O-aminophenoxy)-ethane-N,N,~,~-te~a-acetic acid as extracellular and intracellular Ca2+-chelating agents, respectively, as well as in the presence of the protein-kinase-C inhibitor, H-7. Additionally, genistein, a potent protein-tyrosine kinase inhibitor, was capable of reducing both IgM-stimulated Ca'+ mobilization and p7Tyk activation in a dose-dependent manner. These results indicate that p72'Yk is physically associated with the B-cell-antigen receptor, participating in antigen-mediated signal transduction in both a Ca2+-independent and protein-kinase-C-independent manners.

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We previously demonstrated that thrombin-induced activation of p72syk was independent of intracellular Ca2+ elevation in platelets. However, our previous studies also demonstrated that activation of platelets by ionophore A23187 results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. In the present study, we investigated the effect of Ca2+ elevation on the activity of p72syk. When washed porcine platelets were stimulated with ionophore A23187 and the activity of p72syk was assessed by means of an immunoprecipitation kinase assay, A23187 caused a time- and dose-dependent increase in the specific activity of p72syk. In addition, pretreatment of platelets with both aspirin and ADP scavengers or chelation of extracellular Ca2+ by EGTA had no effect on the A23187-induced activation of p72syk. These results indicate that A23187-induced activation of p72syk is independent of the formation of endoperoxide/thromboxane A2, released ADP and extracellular Ca2+, suggesting the existence of a novel pathway for activation of p72syk. Furthermore, evidence is presented which indicates a synergistic effect of A23187 and thrombin on the activation of p72syk, and an inhibitory effect of pretreatment with phorbol 12-myristate 13-acetate, a protein kinase C activator, on the activation of p72syk induced by either A23187 or thrombin.

We have succeeded in purifying p7Tyk, a non-receptor-type protein-tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991) J. BioE. Chenz. 266, 15 790 -157961 from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellulose, Sephacryl S-200, heparin-Sepharose CL-6B, Mono Q and Mono S. SDSPAGE of the final purified sample revealed a 72-kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross-reactivity with anti-p7Tyk antibody which does not recognize ZAP-70. It was purified approximately 3000-fold with an overall yield of 0.54% according to [ValSIangioten-sin I1 phosphorylation activity and the specific activity of the final sample (30 nmol phosphate .min-' . mg protein-') was relatively lower than that of the 40-kDa kinase, a catalytic fragment of p72yk which lacks two src homology regions 2 domains.The ~7 2 "~ had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [ValSIangiotensin I1 as a substrate, the apparent K,,, value for the peptide was 0.91 mM and that for ATP was 0.48 pM. Mn2+, Mg2+ and Co2+ were effective divalent cations and optimum pH was around 8.0-8.5 for the expression of the activity.These results suggest that the purified p7Tyk may exist as a less active form compared with the 40-kDa kinase and that the part of ~7 2 "~ containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40-kDa kinase.A number of protein-tyrosine kinases (PTK) have been identified so far and can be broadly categorized into two families according to the structure : a transmembrane receptor family and a non-receptor family. The receptor family is believed to initiate the action of the ligand, such as epidermal growth factor, platelet-derived growth factor, insulin and so forth, by binding to it and raising the intrinsic kinase activity. In the case of the non-receptor family, more than ten members have been reported so far, including the syk gene product. However, their function, such as an activation mechanism as well as their position in a signal transduction pathway, is still unclear compared to those of the receptor family [l-51.Previously, we purified a 40-kDa PTK from cytosolic fractions of porcine spleen [6]. Consecutively, the molecular cloning of a gene syk (designated after spleen tyrosine kinase) revealed that it encodes a 72-kDa PTK (p7Tyk) belonging to the non-receptor family and that the 40-kDa kinase was its catalytic fragment generated by proteolysis in the purification procedure. The deduced amino-acid sequence of p72yk has a distinct character from other memb...

Thrombin and epinephrine in combination exert synergistic effects on platelet activation. On the other hand, tyrosine phosphorylation and activation of tyrosine kinases including Syk have been shown to play a critical role in the induction of platelet responses to thrombin stimulation. This study investigated the role of tyrosine phosphorylation and Syk activation in the synergistic mechanisms between thrombin and epinephrine. Although epinephrine alone (4 microM) slightly induced protein-tyrosine phosphorylation and Syk activation, the presence of epinephrine caused a shift to the left in the dose-dependence of thrombin (0.01-0.5 U/ml)-induced tyrosine phosphorylation and Syk activation, as well as platelet aggregation. Phenoxybenzamine, an alpha-adrenoceptor antagonist, canceled this potentiation by epinephrine. Since platelets dominantly express alpha 2-adrenoceptor, this result indicates that epinephrine acts through the occupancy of alpha 2-adrenoceptor. Furthermore, pretreatment with a tyrosine kinase inhibitor, genistein, or a cAMP-elevating agent, prostacyclin (PGI2), significantly reduced these synergistic effects of epinephrine. Taken together, our results suggested that the potentiation by epinephrine may be mediated via enhancement of tyrosine phosphorylation and Syk activation, in part through a decrease of intracellular cAMP levels.