ACTIVATION OF MYELIN BASIC-PROTEIN AND S6 PEPTIDE KINASES IN PHORBOL ESTER-TREATED AND PAF-TREATED SHEEP PLATELETS

The involvement of myelin basic protein (MBP) kinases and ribosomal S6 peptide kinases in sheep platelet signal transduction was investigate d. Treatment of platelets with 200 nM 12-O-tetradecanoylphorbol-13-ace tate (PMA) led to 5-fold stimulations of cytosolic MBP and S6 peptide kinase activities within 1 min. Immunoblotting analysis of phenyl-Supe rose-fractionated cytosol from PMA-treated platelets with a panel of m itogen-activated protein (MAP) kinase anti-peptide antibodies revealed that one of the activated MBP kinases was p42mapk. This MAP kinase is oform was also stimulated to a lesser extent (almost-equal-to 2-fold) when platelets were exposed to 200 muM platelet-activating factor (PAF ) for 3 min. The pathways of PAF-activation of p42mapk also involved a protein kinase C-independent route, since the staurosporin analog com pound 3 reduced PAF-induced activation by almost-equal-to 30% under co nditions in which it inhibited PMA-activation of p42mapk by almost-equ al-to 80%. Another MAP kinase isoform of 44 kDa, most probably p44erk1 , was also detected in platelet cytosol. but it was only marginally mo dulated in response to PMA or PAF. The predominant PMA- and PAF-activa ted MBP kinase detected after MonoQ fractionation of platelet cytosol did not appear to correspond to a MAP kinase. MonoQ chromatography of platelet cytosol also resolved two PMA- and PAF-activated S6 peptide k inases, which appeared to coelute on phenyl-Sepharose. Western blottin g analysis of the MonoQ fractions with antibodies raised against pepti de sequences in the S6 kinases p90rsk and p70S6K revealed immunoreacti ve proteins of almost-equal-to 75 kDa and almost-equal-to 95 kDa that coincided with the first S6 peptide kinase peak. These proteins probab ly corresponded to the 502 and 525 amino-acid-length forms of p70s6k. Only the second peak of S6 peptide kinase activity from MonoQ was appr eciably stimulated in response to PAF-treatment of platelets, and this was largely abolished by compound 3. It is more likely that the novel MBP and S6 peptide kinases described here, rather than p42mapk and p7 0S6K, play a significant role in PAF signal transduction in the platel et.