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.

Protein kinase C ␦ (PKC ␦) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC ␦ in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC ␦ in response to H2O2. P rotein kinase C (PKC) comprises a family of more than ten serine͞threonine protein kinases that are involved in a variety of signal transduction pathways (1). Each isoform has the regulatory and catalytic domains in the amino-and carboxylterminal halves, respectively. The isoforms are divided into three groups, cPKC, nPKC, and aPKC, because of the structural differences in their regulatory domains. The cPKC and nPKC isoforms are activated by diacylglycerol produced from receptormediated hydrolysis of inositol phospholipids and are the prime targets of tumor-promoting phorbol esters that bind to the cysteine-rich sequence, named the C1 region, in the regulatory domain. In general, a number of protein kinases are known to be controlled by phosphorylation (2), and the PKC family members have three phosphorylation motif sites mostly conserved among the family (3). One is a threonine residue in the activation loop of the catalytic domain, and the others are serine and threonine residues located in the carboxyl-terminal end region, named the turn and hydrophobic motifs, respectively.The PKC isoforms are further phosphorylated on tyrosine upon stimulation of the cells (4), and the role of tyrosine phosphorylation has been investigated for PKC ␦, a member of the nPKC group (5). That is, PKC ␦ is phosphorylated on tyrosine in v-ras-transformed keratinocytes (6) and in various cells stimulated with phorbol ester, growth factors, and hormones (7-14). However, controversial results are reported on the functional consequence of the tyrosine phosphorylation reaction induced by these membrane-coupled signaling processes. In keratinocytes, tyrosine phosphorylation reduces its catalytic activity (6, 12), whereas in other cases the modification reaction enhances the enzymatic activity (4, 7, 8) or even alters...

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to as-The LIM domain is a Cys-rich domain composed of 50 -60 amino acid residues with the consensus sequence (Cys-X 2 -Cys-X 17-19 -His-X 2 -Cys) -X 2 -(Cys-X 2 -Cys-X 16 -20 -Cys-X 2 -His/Asp /Cys) (where X represents any amino acid) and is found in various proteins (1, 2): homeodomain-containing transcription factors, cytoskeletal proteins, LIM domain only proteins, protein kinases, and proteins of undefined function. Physicochemical and structural analyses have revealed that the LIM domain is composed of two independent zinc-coordinated fingers (3, 4). Although many zinc finger motifs bind to specific DNA or RNA sequences (5), the LIM domain has been proposed to participate in protein-protein interactions (1, 2). In fact, five proteins have been reported recently as a highly specific target of each LIM domain: the LIM1 domain of zyxin binds to the LIM-only protein CRP by LIM-LIM interaction (6); the CRP forms homodimer by LIM-LIM interaction (7); the LIM domain of RBTN2 binds to the bHLH (basichelix-loop-helix) domain of TAL1 protein (8); the LIM2 and LIM3 domains of Enigma interact with the Tyr-containing tight-turn motifs of the GDNF 1 receptor (GDNFR, known as a Ret Tyr kinase) and the insulin receptor (InsR), respectively (9, 10). Although most LIM domains adopt a similar zinc-coordinated finger consisting of well conserved amino acid sequences, no protein has been identified yet as a common target of LIM domains.The PKC family consists of at least 11 isoforms, which play distinct roles for many cellular functions but show subtle difference of substrate specificities by in vitro phosphorylation studies (11,12). Therefore, it is reasonable to assume that there are some mechanisms by which each PKC isoform recognizes its specific substrate proteins in vivo. Recently, several proteins associating with PKC have been emerged to govern the subcellular localization of the enzyme family (13-16).We report here a novel PKC-binding protein containing three LIM domains, designated ENH, and show the association of PKC with LIM domains of different proteins including this novel PKC-binding protein, suggesting that protein-protein interaction with PKC is a general property of LIM domains. EXPERIMENTAL PROCEDURESYeast Two-Hybrid Assay-The yeast two-hybrid assay (17) was conducted in the yeast strain CG-1945 , a derivative of HF7c (18), by using a fusion between GAL4 DNA binding domain and the regulatory domain of rat PKC ␤I (residues 1-340) (19) as a bait. ␤-Galactosidase activity in yeast cells was measured by plate assay methods. All measurements were repeated at least four times.Expression of Epitope-tagged ENH in COS-7 Cells-We constructed two parental vectors, pTB701-FLAG and pTB701-HA, for expression o...

Redox polymer layers with 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl (TEMPO) groups showed nernstian adsorbate-like electrochemical behaviors up to submicrometer thicknesses, based on a fast charge propagation within the bulk layer and persistency in electrolyte solutions.