ELSEVIER Neuroscience Letters 214 (1996) 13-16 lml H7, an inhibitor of protein kinase C, prevents serum-induced phosphorylation of Raf and MAP kinase in neuroblastoma cells Hugo Cabedo, Vicente Felipo*, Maria-Dolores Mifiana, Santiago Grisolia lnstituto de lnvestigaciones Citolfgicas de la Fundaci6n Valenciana de lnvestigaciones BiomOdicas, Amadeo de Saboya, 4, 46010 Valencia, Spain Received 10 June 1996; revised version received 28 June 1996; accepted 28 June 1996 Abstract We have previously shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, inhibits proliferation of neuroblastoma cells in culture. We have now tested whether the effect of H7 is mediated by MAP kinase and Raf. It is shown that, in Neuro 2a cells, activation of protein kinase C by addition of 4~3-phorbol-12/3-myristate-13~-acetate(PMA), leads to phosphorylation of Raf and Mitogen-activated protein kinase (MAP kinase). PMA-induced phosphorylation of these proteins is pre- vented by H7. When quiescent Neuro 2a were stimulated to proliferate by addition of serum, Raf and MAP kinase were rapidly phosphorylated. Serum-induced phosphorylation of Raf and MAP kinase is prevented by H7. These results suggest that, in Neuro 2a cells, the control of proliferation by protein kinase C could be mediated by phosphorylation (and concomitant activation) of Raf and MAP kinase. Keywords: Protein kinase C; MAP kinase; Raf; H7; Neuroblastoma; Cell proliferation Protein kinase C (PKC) plays an important role in the control of cell proliferation. The involvement of PKC in the control of cell growth was initially suggested by the finding that PKC constitutes the receptor for mitogenic phorbol esters, which are potent tumour promoters[12]. We have previously shown that 1-(5-isoquinolinylsulfo- nyl)-2-methylpiperazine (H7), a non-specific inhibitor of PKC, prevents proliferation of neuroblastoma cells (Neuro 2a) in culture [7,13,14], of other cell lines [15,16] and of primary cultured cells from human brain tumours [ 15]. H7 also inhibits tumour cell division in mice bearing ascitic Ehrlich's carcinoma [8]. Moreover, PKC activity is increased in some human tumours [9]. These facts support the idea that PKC plays a key role in the control of cell proliferation. Mitogen-activated protein kinases (MAP kinases), also described as extracellular signal-regulated kinases (ERKs), belong to a group of protein-serine/threonine kinases that are activated in response to various stimuli (growth factors, neurotransmitters, etc.). It has been reported that MAP kinases are involved in the control of * Corresponding author. Tel.: +34 6 3698500; fax: +34 6 3601453. cell proliferation [3,5,17]. A unique feature of this family of protein kinases is that they require dual phosphorylation on both tyrosine and threonine residues to become fully active [2]. It has been shown that activation of PKC can lead to phosphorylation and activation of MAP kinase [1,10]. It was therefore considered of interest to test whether the effect of H7 on Neuro 2a cell proliferation is mediated by alterations in MAP kinase. 4/3-Phorbol-12/3-myristate-13cc-acetate (PMA) was from Molecular Probes (Eugene, OR, USA). H7 was from Sigma (St. Louis, MO, USA). The clonal cell line Neuro 2a, C1300 mouse neuroblas- toma, was obtained from the American Type Culture Col- lection. Cells were grown at 37°C in Eagle's minimum essential medium (MEM) supplemented with 10% foetal bovine serum, 100 IU of penicillin per ml, and 100 mg/ml of streptomycin. For synchronization, the cells were seeded (400000 cells/ml) in MEM without glutamine containing 0.5% foe- tal bovine serum and antibiotics as above; after 48 h the cells were in Go. To stimulate cell proliferation the med- ium was removed, the cells were washed and stimulated by addition of complete MEM with 10% foetal bovine serum. 0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12867-6