Expression of GTP-binding proteins and protein kinase C isozymes in platelet-like particles derived from megakaryoblastic leukemia cells (MEG-01).
The expression of the various GTP-binding proteins and protein kinase C (PKC) in the platelet-like particles produced by MEG-01 cells was analyzed by RT-PCR and immunoblotting. We selected 14 human low Mr GTPbinding proteins (LMW-GPs) and nine PKCs expressed in platelets and/or megakaryocytes, and designed specific primer pairs for the proteins. RT-PCR analysis revealed that the particles express the mRNAs of many LMW-GPs such as rap 1A, rap 1B, rap 2B, ral A, rho A, rac 1, rac 2, Cdc 42, rab 1, rab 3B, rab 6, ram/rab 27 and ran . By immunoblotting analysis, Rap1, RhoA, Rac, Cdc42, Rab6 and Rab8 were identified in the particles. As for PKCs, the particles were observed to express the mRNAs of PKC-alpha,-beta I, -beta-II, -delta, epsilon, eta and theta, but not-gamma and zeta. Using immunoblot analysis, PKC-beta I, -beta II and zeta were shown to exist in the particles, although the contents were lower than those in platelets. Furthermore, the presence of Gi2-alpha, a heterotrimeric G protein that is the major pertussis toxin substrate in human platelets, and beta subunits was observed in the particles. Taken together, the particles possess some similarity to human platelets based on the expression profiles of GTP-binding proteins and PKCs.
Nagata
K
K
Department of Molecular Pathobiochemistry, Gifu University School of Medicine, Japan. kinagata@cc.gifu-u.ac.jp
Okano
Y
Y
Nozawa
Y
Y