Protein kinase C (PKC) isoforms, ␣, ␤I, and ␥ of cPKC subgroup, ␦ and of nPKC subgroup, and of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H 2 O 2 . These isoforms isolated from the H 2 O 2 -treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC ␣ and ␦, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC ␦ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H 2 O 2 . These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr 308 and Ser 473 ) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ϳ75% in CHO cells and ϳ30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4,14,25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11,23). These observations indicate that Akt is a downstream effector of PI 3-kinase.PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6,16,31,39,61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase ...

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.Akt is a protein serine-threonine kinase that contains a pleckstrin homology domain and whose kinase domain has structural similarity with those of protein kinase C (PKC) isozymes and cyclic AMP (cAMP)-dependent protein kinase (PKA) (9, 21). Thus, Akt has also been termed protein kinase B. Akt was originally shown to be activated by growth factors such as platelet-derived growth factor and insulin, but later the enzyme was also found to be activated by cytokines and ligands for G protein-coupled receptors (21,33,34). Moreover, expression of polyomavirus middle T antigen as well as cellular stresses such as hyperosmolarity, heat shock, and fluid shear stress also induces activation of Akt (17,27,42). However, the mechanisms by which Akt is activated by these diverse stimuli are not fully understood. The activation of Akt by growth factors or cytokines is blocked by pharmacological or molecular biological inhibitors of phosphoinositide (PI) 3-kinase (7,19,24), indicating that PI 3-kinase is an upstream regulator of Akt, although PI 3-kinase-independent stimuli that induce activation of Akt also appear to exist (27,33,38).Akt is a general mediator of cell survival and protection from apoptosis (9, 21). It has also been suggested to participate in meiosis in oocytes (3), in endocytosis elicited by RAS (5), in differentiation of adipocytes (25), and in various metabolic actions of insulin (23,25,44,45). In spite of the potential importance of Akt in such diverse biological activities, only a few proteins have been identified as physiological substrates of this enzyme. The first iden...

A nitroxide radical functional polymer was photocrosslinked for the first time without significant side reactions, producing a cathode-active thin film, leading to an organic-based paper battery.

The present results demonstrate that a sufficient posterior shift of the spinal cord and neurologic improvement will not be obtained after posterior decompression surgery in the K-line (-) group. Our new index, the K-line, is a simple and practical tool for making decisions regarding the surgical approach for cervical OPLL patients.