The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

Potent induction of wild-type p53-dependent transcription in tumour cells by a synthetic inhibitor of cyclin-dependent kinases.

Activation of the p53 tumour suppressor protein by distinct forms of stress leads to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. The cyclin-dependent kinase inhibitor roscovitine has been shown to induce nuclear accumulation of wild-type p53 in human untransformed and tumour-derived cells. We analyzed the response of different human tumour cell lines to roscovitine treatment with respect to their p53 status. Striking induction of wild-type p53 protein and dramatic enhancement of p53-dependent transcription, coinciding with p21WAF1 induction, was observed in wildtype, but not mutant, p53-bearing tumour cells after treatment with roscovitine. The transcriptional activity of p53 was substantially higher in roscovitine-treated cells than in cells irradiated with ultraviolet C or ionizing radiation, even though all these agents induced a similar amount of p53 accumulation. These results highlight the therapeutic potential of roscovitine as an anticancer drug, especially in tumours retaining a functional wild-type p53 pathway.