In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system.

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.

Phosphorylation of the fused protein kinase in response to signaling from hedgehog.

The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins.

We have isolated an Arabidopsis thaliana gene that codes for a receptor related to antifungal pathogenesis-related (PR) proteins. The PR5K gene codes for a predicted 665-amino acid polypeptide that comprises an extracellular domain related to the PR5 proteins, a central transmembrane-spanning domain, and an intracellular protein-serine/threonine kinase. The extracellular domain of PR5K (PR5-like receptor kinase) is most highly related to acidic PR5 proteins that accumulate in the extracellular spaces of plants challenged with pathogenic microorganisms. The kinase domain of PR5K is related to a family of protein-serine/threonine kinases that are involved in the expression of self-incompatibility and disease resistance. PR5K transcripts accumulate at low levels in all tissues examined, although particularly high levels are present in roots and inflorescence stems. Treatments that induce authentic PR5 proteins had no effect on the level of PR5K transcripts, suggesting that the receptor forms part of a preexisting surveillance system. When the kinase domain of PR5K was expressed in Escherichia coli, the resulting polypeptide underwent autophosphorylation, consistent with its predicted enzyme activity. These results are consistent with PR5K encoding a functional receptor kinase. Moreover, the structural similarity between the extracellular domain of PR5K and the antimicrobial PR5- proteins suggests a possible interaction with common or related microbial targets.

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.

Molecular cloning of a protein kinase whose phosphorylation is regulated by genetic adhesion during Chlamydomonas fertilization.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine beta-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.