HORMONAL REGULATION OF CELLULAR METABOLISM

蛋白质的磷酸化-去磷酸化是其中最重要的一个

Phosphorylation-dephosphorylation of proteins is one of the most important mechanisms for the regulation of cellular functions. Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, has emerged as a pivotal regulatory element for cell growth, differentiation, gene expression, hormone secretion, cell surface receptor function, and cellular metabolism. This protein kinase can be activated by diacylglycerol, a second messenger generated by signal-induced breakdown of phosphoinositides. In addition, it has been identified as a receptor for tumor-promoting phorbol esters which elicit pleiotropic responses comparable to those stimulated by many hormones and growth factors. Three isozymic forms of PKC have been identified from mammalian brains. Polyclonal and monoclonal antibodies against these enzymes were prepared for their immunochemical characterization. These enzymes were found to have distinct tissue, cellular, and subcellular distributions and were differentially expressed during development. The role of PKC in mediating the action of neurotransmitter was investigated with neurons grown in culture. These cells express PKCII and III in a similar growth-regulated pattern as in intact cerebella of postnatal rats. Stimulation of the cultured granule cells with the excitatory amino acid, glutamate, or phorbol ester causes the association of both PKCs with the particulate fraction; however, only PKCII, but not PKCIII, was found to associate with the membrane skeleton. Membrane association of PKC in the cell is an obligatory step for activation. Several inositol-containing phospholipids were found to interact with PKC with high affinity, in particular, the CNS-specific PKCI. Interaction of PKC with these phospholipids places the enzyme in close proximity to diacylglycerol generated by the activation of phospholipase C. This class of phospholipids may serve as anchoring sites of PKC at the membrane. Activation of PKC in the control of gene expression was investigated by studying the phosphorylation of a transcription factor, CCAAT/enhancer-binding protein (CEBP), which contains a leucine-repeat dimerization interface and an upstream basic region for DNA binding. Phosphorylation of a truncated form of CEBP at two Ser residues within the basic region results in attenuation of DNA binding. Mutation of one of the Ser residues to Cys obviates the PKC-mediated response, suggesting a role of PKC in transcriptional control. The genomic structure of PKC I gene was analyzed for the purpose of defining tissue-specific and development-regulated expression of this kinase. A genomic fragment containing the 5'-flanking region, the first exon, and the first intron, has been isolated and sequenced. Several potential transcriptional factor binding sites have been identified by footprint analysis.