Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.     

Spermatogenesis in Bclw-deficient mice.

Bclw is a death-protecting member of the Bcl2 family of apoptosis-regulating proteins. Mice that are mutant for Bclw display progressive and nearly complete testicular degeneration. We performed a morphometric evaluation of testicular histopathology in Bclw-deficient male mice between 9 days postnatal (p9) through 1 yr of age. Germ cell loss began by p22, with only few germ cells remaining beyond 7 mo of age. A complete block to elongated spermatid development at step 13 occurred during the first wave of spermatogenesis, whereas other types of germ cells were lost sporadically. Depletion of Sertoli cells commenced between p20 and p23 and continued until 1 yr of age, when few, if any, Sertoli cells remained. Mitochondria appeared to be swollen and the cytoplasm dense by electron microscopy, but degenerating Bclw-deficient Sertoli cells failed to display classical features of apoptosis, such as chromatin condensation and nuclear fragmentation. Macrophages entered seminiferous tubules and formed foreign-body giant cells that engulfed and phagocytosed the degenerated Sertoli cells. Leydig cell hyperplasia was evident between 3 and 5 mo of age. However, beginning at 7 mo of age, Leydig cells underwent apoptosis, with dead cells being phagocytosed by macrophages. The aforementioned cell losses culminated in a testis-containing vasculature, intertubular phagocytic cells, and peritubular cell "ghosts." An RNA in situ hybridization study indicates that Bclw is expressed in Sertoli cells in the adult mouse testis. Consequently, the diploid germ cell death may be an indirect effect of defective Sertoli cell function. Western analysis was used to confirm that Bclw is not expressed in spermatids; thus, loss of this cell type most likely results from defective Sertoli cell function. Because Bclw does not appear to be expressed in Leydig cells, loss of Leydig cells in Bclw-deficient mice may result from depletion of Sertoli cells. Bclw-deficient mice serve as a unique model to study homeostasis of cell populations in the testis.     

Lack of regulation of aromatic L-amino acid decarboxylase in intact bovine chromaffin cells

Aromatic l-amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate-limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32P(i)) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.     

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.     

Multiple site phosphorylation of tyrosine hydroxylase. Differential regulation in situ by a 8-bromo-cAMP and acetylcholine

Suspension cultures of purified bovine adrenal chromaffin cells incorporated 32P from exogenous 32Pi into a protein of approximately M4 = 60,000 (isolated by discontinuous, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis). Phosphorylated tyrosine hydroxylase, purified from chromaffin cell supernatants by immunoprecipitation, co-migrated with the Mr = 60,000 band. Tryptic fragments prepared fom either the Mr congruent to 60,000 band or the immunoprecipitated tyrosine hydroxylase band were analyzed after separation with two-dimensional electrophoresis/chromatography. Two distinct 32P-peptides were present in either sample. After a 2-3-min lag period. 32P incorporation into both peptides was relatively linear with time for at least 20 min. In the presence of calcium, exogenous acetylcholine (100 microM) increased 32P incorporation into both of the 32P-labeled tryptic peptides whereas 8-bromo-cAMP (1 mM) increased 32P incorporation into only one of the two. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and MnCl2 inhibited the acetylcholine-induced phosphorylation of both tryptic peptides. Thus, tyrosine hydroxylase is phosphorylated in situ at more than one site, and the phosphorylation of these sites is affected differently by acetylcholine and 8-bromo-cAMP. The data imply that kinase activity other than (or in addition to) cAMP-dependent protein kinase activity attends tyrosine hydroxylase in the intact chromaffin cells and that multiple kinase activities may be involved in the short term regulation of catecholamine biosynthesis by afferent activity.     

Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene

Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta-galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress.     

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronai elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.Abbreviations EPT ethanolaminephosphotransferase - CPT cholinephosphotransferase - NMT N-methyltransferase This work supported by funds provided to the Section of Pediatric Neurology by Texas Children's Hospital.