Although the involvement of protein kinase C (PKC) in the activation of the mitogen-activated protein (MAP) kinase pathway has been implicated through experiments using 12-O-tetradecanoylphorbol-13-acetate (TPA), there has been no direct demonstration that PKC activates the MAP kinase pathway. A Raf-dependent intact cell assay system for monitoring the activation of MAPK/ERK kinase (MEK) and extracellular signal-related kinase (ERK) permitted us to evaluate the role of PKC isotypes in MAP kinase activation. Treatment of cells with TPA or epidermal growth factor resulted in the activation of MEK and ERK. The activation of the MAP kinase pathway triggered by epidermal growth factor was completely inhibited by dominant-negative Ras (RasN17), whereas the activation triggered by TPA was not, consistent with previous observations. The introduction of an activated point mutant of PKCdelta, but not PKCalpha or PKCepsilon, resulted in the activation of the MAP kinase pathway. The activation of MEK and ERK by an activated form of PKCdelta requires the presence of c-Raf and is independent of RasN17. These results demonstrate that activation of PKCdelta is sufficient for the activation of MEK and ERK and that the pathway operates in a manner dependent on c-Raf and independent of Ras.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKCO, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC8 (67.0% in total). nPKCO has a zinc-finger-like cysteine-rich sequence (Cl region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKCO lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKCa, -13I, -1311, and -y) but not in the novel PKC subfamily (nPKC8, -e, -C, and -i). Northern (RNA) blot analyses revealed that the mRNA for nPKCO is expressed predominantly in skeletal muscle. Furthermore, nPKCO mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKCO expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKCO sequence, nPKCO was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKCO cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKCO was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKCO should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.Protein kinase C (PKC) plays a key regulatory role in a variety of cellular functions, such as cell growth and differentiation, gene expression, secretion of hormones and neurotransmitters, and membrane functions (29,30). PKC was originally identified as a serine/threonine protein kinase whose activity was dependent on calcium and phospholipids (55). Diacylglycerols (DAG), products of phosphatidylinositol turnover, lower the Ca + requirement for PKC activity from millimolar to micromolar concentrations in the presence of phosphatidylserine (PS) (18, 56), indicating that PKC is a target of DAG. Tum6r-promoting phorbol esters also bind to and activate PKC (5, 15). The pleiotropic action of phorbol esters could be explained as mimicking the activity of PKC by substituting for a physiological PKC activator, DAG. Besides the classical pathway of DAG generation through receptor-mediated hydrolysis of inositol phospholipids, recent experiments have revealed the presence of a novel pathway of DAG generation through hydrolysis of phosphatidylcholine (PC) (9). It was suggested that PKC is also involved in signaling pathways that generate DAG from PC.Recent molecular cloning experiments have established that PKC molecules consist of a protein family which can be classified into at least two major classes, conventional PKC (cPKC; isoforms a, 13I, ,II, and -y) and novel PKC (nPKC; isoforms 8, e, and t)

Overexpression of a TPA‐insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet‐derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal‐dependent changes in aKPClambda were observed for a PDGF receptor add‐back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3‐kinase, and were inhibited by wortmannin, an inhibitor of PI3‐kinase. Furthermore, an N‐terminal fragment of the catalytic subunit of PI3‐kinase, p110alpha, inhibited aPKClambda‐dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3‐kinase.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by theXenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of theXvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.