Coordinated regulation of Ras-, Rac-, and Ca2+-dependent signaling pathways.

Stimulation of cells by a broad variety of agonists results in a coordinated activation of Ca2+-, Ras-, and Rac-dependent pathways, which have to synergize to yield the corresponding biological response. This synergy requires a dense net of mutually positive and negative regulatory mechanisms. These include Ca2+-mediated activation of guanine nucleotide exchange proteins, resulting in increased levels of GTP-charged Ras and/or Rac or inactivation of small GTPases by Ca2+-mediated activation of GTPase-activating enzymes. Likewise, Ras as well as Rac control regulatory mechanisms, resulting in increased or diminished levels of cytosolic-free Ca2+. The biochemical mechanisms underlying these effects are discussed, with special reference to regulation of Ca2+ signaling by Ras and Rac.

Critical role of protein kinase C alpha and calcium in growth factor induced activation of the Na(+)/H(+) exchanger NHE1.

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.

Defining the human targets of phorbol ester and diacylglycerol.

Phorbol esters (PEs) and their derivatives are potent tumor-promoting agents. The best known receptors for these substances are the novel and classical isotypes of protein kinase C (PKC), which bind PE and the physiological second messenger diacylglycerol (DAG) by cysteine-rich domains, the C1 domains. However, PKC is not the sole receptor of PE, a concept that has been largely ignored in the past. PE (in addition to DAG) also targets C1-containing receptors unrelated to PKC. In order to get a better insight into DAG/PE-mediated signaling and the pathways involved, it is necessary to first determine all ligand-interacting proteins. Employing various sources of data, 66 different C1-containing human proteins are presented and predictions of their DAG/PE-binding potential are attempted. Defining the entire set of key mediators for the physiological DAG responses and for PE-induced tumorigenesis may aid our understanding of signal integration and can also help to design new strategies for therapeutic cancer intervention.