Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

The(1) regulatory mechanism of glucose uptake in 3T3-L1 adipocytes was investigated with the use of recombinant adenovirus vectors encoding various dominant negative proteins. Infection with a virus encoding a mutant regulatory subunit of phosphoinositide (PI) 3-kinase that does not bind the 110-kDa catalytic subunit (delta p85) inhibited the insulin-induced increase in PI 3-kinase activity co-precipitated by antibodies to phosphotyrosine and glucose uptake in a virus dose-dependent manner. Overexpression of a dominant negative RAS mutant in which Asp57 is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant that lacks guanine nucleotide exchange activity (delta SOS) abolished the insulin-induced increase in mitogen-activated protein kinase activity, but had no effect on PI 3-kinase activity or glucose uptake. Although GH and hyperosmolarity attributable to 300 mM sorbitol each promoted glucose uptake and translocation of glucose transporter (GLUT)4 to an extent comparable to that of insulin, these stimuli triggered little or no association of PI 3-kinase activity with tyrosine-phosphorylated proteins. Overexpression of delta p85 or treatment of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no effect on glucose uptake or translocation of GLUT4 stimulated by GH or hyperosmolarity. Moreover, overexpression of delta SOS or RAC17N also did not affect the increase in glucose uptake induced by these stimuli. A serine/threonine kinase Akt, a constitutively active mutant of which was previously shown to stimulate glucose uptake, is activated by insulin, GH, and hyperosmolarity to approximately 4-fold, approximately 2.1-fold, and approximately 2.3-fold over basal level, respectively. These results suggest that insulin-induced but neither GH- or hyperosmolarity-induced glucose uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulininduced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c −/−

The involvement of the phosphatidylinositol 3-kinase pathway in the regulation of melanogenesis was examined using human G361 melanoma cells. In the cells treated with wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, the melanin content increased concomitant with the elevated protein level of tyrosinase, a key enzyme in melanogenesis. Northern blot analysis revealed that the mRNA level of tyrosinase increased transiently on treatment of the cells with the phosphatidylinositol 3-kinase inhibitor. When the cells were infected with the adenovirus vector encoding the mutant adapter subunit of phosphatidylinositol 3-kinase, which acts as a dominant negative of phosphatidylinositol 3-kinase, both the melanin content and the expression of tyrosinase increased. In cells infected with the adenovirus vector encoding the constitutively active mutant of the lipid kinase, a decrease in melanin content as well as reduced expression of tyrosinase was observed. In cells expressing the constitutively active mutant of the serine-threonine protein kinase Akt, one of the downstream targets of phosphatidylinositol 3-kinase, the melanin content decreased as in the cells overproducing the constitutively active mutant of phosphatidylinositol 3-kinase. These results indicate that phosphatidylinositol 3-kinase regulates melanogenesis by modulating the expression of tyrosinase, and that activation of Akt is sufficient for suppression of melanin production in G361 melanoma cells.

Steroid hormones modulate physiological processes by a number of mechanisms including regulation of gene expression. We wondered if glucocorticoids might induce expression of al adrenergic receptors, which could contribute to the increased sensitivity of vascular smooth muscle to catecholamines that may occur with glucocorticoid excess. We examined the effects of dexamethasone on the expression of the alB adrenergic receptor gene in DDT1 MF-2 smooth muscle cells. Dexamethasone (10' M) produced a 1.8±0.2-fold increase in expression of alB receptors determined with VI-Hprazosin. Steady-state values of aiB adrenergic receptor mRNA, analyzed by Northern blotting, increased 2.8±0.7-fold after 48 h exposure to dexamethasone. This effect of dexamethasone occurred in the presence of the protein synthesis inhibitor cycloheximide. alB receptor mRNA abundance was also increased by testosterone and aldosterone, whereas f estradiol and progesterone had no effect. The a1B receptor gene transcription rate, determined in nuclear run-off assays, increased 2.6±0.6-fold in cells treated with dexamethasone for 24 h. The half-life of the alB receptor mRNA was unchanged by dexamethasone. These data indicate that glucocorticoids regulate expression of aiB receptors by increasing the rate oftranscription ofthis gene. (J. Clin. Invest.