PMA‐induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level

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Abstract
We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10−7 m phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (α, βI, βII, γ, δ, ε, θ, η, ζ) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant (P < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC-α, -βI, -ε, -θ and -ζ isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-ζ isoforms. Moreover, PKC-δ appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10−7 m haemin. The possible implications of these findings for a better understanding of the molecular events underlying the process of megakaryocytic differentiation are discussed.

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