Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

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.

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3' untranslated regions of human alpha-tropomyosin.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).