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

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 (alpha, beta I, beta II, gamma, delta, epsilon, theta, eta, zeta) 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-alpha, -beta I, -epsilon, theta and -zeta isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-zeta isoforms. Moreover, PKC-6 delta 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.

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

“…Human erythroleukemia (HEL) cells grown in suspension can be differentiated to adherent megakaryocytes by phorbol 12-myristate 13-acetate (PMA) treatment, providing an opportunity to analyze how the profile of sialoglycoproteins changes upon differentiation with various labeling methods (13)(14)(15). Metabolic labeling of HEL cells with Ac 4 ManNAz showed predominantly O-sialoglycoproteins being labeled as demonstrated by the insensitivity of labeled glycoproteins to PNGase F treatment (Fig.…”

Integrin Leukocyte Function-associated Antigen-1-mediated Cell Binding Can Be Activated by Clustering of Membrane Rafts

“…The induction of thymocyte binding with mAb to CD24 or CTx was blocked by the PI3-kinase inhibitor wortmannin and the Tyr kinase inhibitor genistein. PI3-kinase has been implicated in the activation of the ␣ IIb ␤ 3 integrin of platelets and megakaryocytes (35) and appears to be involved in phosphorylation events during cytoskeletal reorganization, growth factordependent mitogenesis, prevention of apoptosis, cytokine production, and endocytic trafficking (36 -38). Recent studies have implicated PI3-kinase in the regulation of ␤2-integrin-mediated adhesion of Jurkat T cells to ICAM-1 (39,40).…”

Thrombopoietin-induced conformational change in p53 lies downstream of the p44/p42 mitogen activated protein kinase cascade in the human growth factor-dependent cell line M07e

“…The role MAPK plays with regard to apoptosis suppression certainly appears to be dominant to the role PI-3K plays in this cytokine/cell system. PI-3K may therefore supplement the anti-apoptotic activities of MAPK while serving another more vital function such as adhesion or proliferation induction (Dorsch et al, 1997;Takahira et al, 1997;Zauli et al, 1997).…”

“…Some reports have demonstrated the ability of Tpo to activate PI-3K, which has been shown to suppress apoptosis in some cell systems (Pa rrizas et al, 1997;Sattler et al, 1997;Zauli et al, 1997). We therefore tested the involvement of PI-3K in p53 conformational change and Tpo-enhanced viability in M07e cells using wortmannin, a well-characterized inhibitor of PI-3K activity that we have previously shown to be biologically eective at a signi®cant level in this cell line Takahira et al, 1997).…”