A human megakaryoblastic cell line, CMK, was treated with 12-o-tetradecanoylphorbo 1-13-acetate (TPA) for differentiation-induction. We examined TPA-induced activation of the MEK1-ERKI/2 pathway in the 100,000g Triton X-insoluble fraction of CMK cells as the membrane skeleton and researched the relation of the MEK1-ERK1/2 activation with integrin expression. We found that this activation was divided into two phases: the first activation occurred transiently in the membrane skeleton fraction of the suspended cell status and diminished after I h; and the second sustained activation was maintained by cell adhesion. TPA-treated CMK cells revealed increased expression of integrins alphaIIb and P3 only when the cell adhesion persisted, regardless of the difference of culture substratum. Sustained activation of the MEK10-ERK1/2 pathway is generated in the membrane skeleton by continuous cell adhesion and seems to be essential to TPA-induced megakaryocytic differentiation of CMK cells. (C) 2002 Elsevier Science (USA). All rights reserved.