Oda A, Druker B J, Ariyoshi H, Smith M, Salzman E W
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02115.
J Biol Chem. 1993 Jun 15;268(17):12603-8.
Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may have regulatory effects on the kinase.
钙蛋白酶几乎在所有组织中都普遍存在（克罗尔，D. E.，和德马蒂诺，G. N.（1991年）《生理学评论》71卷，813 - 846页），但其生理作用仍有待确定。鉴定其天然内源性底物将非常有意义。由于血小板中的主要酪氨酸激酶pp60src在从细胞中纯化过程中已知容易被切割（费德，D.，和毕晓普，J. M.（1990年）《生物化学杂志》265卷，8205 - 8211页），我们研究了它是钙蛋白酶内源性底物的可能性。在用单克隆抗体327进行蛋白质印迹分析的静息血小板全细胞裂解物中，我们发现pp60src几乎完全以60 kDa的形式存在，有微量的52 kDa形式。添加已知为血小板钙蛋白酶激活剂的A23187（一种钙离子载体）或丁卡因（克罗尔和德马蒂诺，1991年；福克斯，J. E.，雷诺兹，C.，莫罗，J. S.，和菲利普斯，D. R.（1987年）《血液》76卷，2510 - 2519页；福克斯，J. E.，奥斯汀，C. D.，博伊尔斯，J. K.，和斯特芬，P. K.（1990b年）《细胞生物学杂志》111卷，483 - 493页），导致肌动蛋白结合蛋白和p235蛋白（踝蛋白）呈剂量和时间依赖性切割。与此同时，通过蛋白质印迹检测到60 kDa的pp60src种类减少，52 kDa（偶尔表现为双峰）和47 kDa种类产生。在由1单位/毫升凝血酶聚集的血小板中，发现了明显相同的切割产物。pp60src的切割被钙蛋白酶抑制剂钙肽素（20 microM）抑制（辻仲中，T.，梶原，Y.，神林，J.，酒井，M.，樋口，N.，田中，T.，和森，T.（1988年）《生物化学与生物物理研究通讯》153卷，1201 - 1208页；辻仲中，T.，有吉，H.，上村，Y.，酒井，M.，神林，J.，和森，T.（1990年）《生命科学》46卷，1059 - 1066页；福克斯，J. E.，克利福德，C. C.，和奥斯汀，C. D.（1990年）《血液》76卷，2510 - 2519页；福克斯，J. E.，奥斯汀，C. D.，博伊尔斯，J. K.，和斯特芬，P. K.（1990年）《细胞生物学杂志》111卷，483 - 493页；福克斯，J. E.，奥斯汀，C. D.，克利福德，C. C.，和斯特芬，P. K.（1991年）《生物化学杂志》266卷，13289 - 13295页）。向细胞外培养基中添加EGTA（3 mM）完全抑制了响应A23187（1 microM）时肌动蛋白结合蛋白、踝蛋白和pp60src的切割。完整的pp60src分布在细胞质和颗粒（膜）部分。切割后的种类仅在细胞质部分被发现。与pp60src相关的烯醇化酶激酶活性降低。因此，pp60src是钙蛋白酶的内源性底物，其切割可能对激酶具有调节作用。
