No abstract

Calmodulin (CaM) is a ubiquitous Ca(2+) receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.

(ii) proteolysis of four out of the five enzymes in the absence of calmodulin activates their respective enzymic activities (Table 2). In fact, earlier studies revealed that Vol.

A ligand-insensitive form of the human epidermal growth factor receptor (EGFR) was enriched by Ca2+-dependent calmodulin-affinity chromatography purification. The basic amphiphilic segment Arg645-Arg-Arg-His-Ile-Val-Arg-Lys-Arg-Thr654-Leu-Arg-Arg-Le u-Leu-Gln 660, located within the cytoplasmic juxtamembrane domain of this receptor, was purified as a fusion protein with glutathione S-transferase and shown to bind calmodulin in a Ca2+-dependent manner. An apparent dissociation constant of 0.4 microM calmodulin (Kd'(CaM)) and an apparent affinity constant of 0.5 microM free Ca2+ (Ka'(Ca)) were measured for this binding process. Binding of calmodulin at the juxtamembrane site prevented the phosphorylation of residue Thr-654 by protein kinase C, and an apparent inhibition constant of 0.5-1 microM calmodulin (Ki'(CaM)) was determined. Conversely, phosphorylation of this site by protein kinase C prevented its subsequent interaction with calmodulin. We therefore propose that cross talk between signaling pathways mediated by calmodulin and protein kinase C occurs at the juxtamembrane domain of the EGFR. This calmodulin-binding sequence is highly conserved among protein tyrosine kinases of the vertebrate EGFR family.

Nitric oxide (NO•) has been proposed to be a physiological modulator of cell proliferation, able to promote in most cases cell cycle arrest. In this review I explore the molecular basis of this mechanism of action. The modulatory action of NO• on the intracellular concentration of cGMP and the machinery directly involved in the control of cell cycle progression, including the expression and activity of diverse cyclins and cyclin‐dependent kinases, their physiological inhibitors, and the master transcriptional regulator retinoblastoma protein, will be discussed. The role of NO• in proliferation mediated by tyrosine kinase receptors such as the epidermal growth factor receptor and downstream signalling pathways will also be considered. Finally, the involvement of NO• in proliferative processes relevant for normal development will be outlined.

No abstract

Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl}propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor.

Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.Keywords: calmodulin; calmodulin-dependent systems; cellular signalling; phosphocalmodulin; protein kinases; phosphoprotein phosphatases. I N T R O D U C T I O N Calmodulin as a Ca 2+ sensorThe average cytosolic concentration of free Ca 2+ in resting cells ranges from 20 to 50 nM, reaching values close to 1 lM when the cell is stimulated by a variety of physiological stimuli, while in the extracellular milieu this concentration is about 1 mM. This large concentration gradient allows intracellular Ca 2+ to work as an useful second messenger [1,2]. The cytosolic Ca 2+ concentration is exquisitely regulated by the operation of transport systems responsible for its increase, represented by different types of Ca 2+ channels and Na + (H + )/Ca 2+ exchangers located in the plasma membrane, the endo(sarco)plasmic reticulum, and/or the mitochondria; and extrusion transport systems represented by Ca 2+ -ATPases located in both the plasma membrane and the endo(sarco)plasmic reticulum, and the Na + /Ca 2+ exchanger also located within the plasma membrane [1,3]. The operation of theses transporters gives rise to oscillations in the concentration of Ca 2+ not only in the cytosol but in the nucleus and intracellular organelles [1,4,5]. It has been possible to observe in living cells inhomogeneities in these transient changes inCorrespondence to A. Villalobo, Instituto de Investigaciones Biome´dicas, Consejo Superior de Investigaciones Cientı´ficas and Universidad Auto´noma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain. E-mail: antonio.villalobo@iib.uam.es Abbreviations: AdeCycl, adenylate cyclase; CaM, calmodulin; CaM-BD, calmodulin-binding domain; CaMPK-II, calmodulin-dependent protein kinase II; CK-II, casein kinase II; CKR,...