Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.

Interleukin 1 is the prototype of an inflammatory cytokine, and evidence suggests that it uses the sphingomyelin pathway and ceramide production to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinated photoaffinity labeling analog of ceramide ([125I] 3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramide) was employed. It is observed that ceramide specifically binds to and activates protein kinase c-Raf, leading to a subsequent activation of the MAPK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protein kinase C-zeta. These data identify protein kinase c-Raf as a specific molecular target for interleukin 1 beta-stimulated ceramide formation and demonstrate that ceramide is a lipid cofactor participating in regulation of c-Raf activity.

Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors.

A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases. One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources. Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution. The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases. Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring. We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8. Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents.

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.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve.

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.

Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

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.

Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages.

Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, we found that LPS stimulated AP-1-mediated transcription of a transfected reporter gene in the murine macrophage cell line RAW 264.7. As AP-1 activity is regulated in part by activation of the c-Jun N-terminal kinase (JNK), which phosphorylates and subsequently increases the transcriptional activity of c-Jun, we examined whether LPS treatment of macrophages resulted in activation of this kinase. LPS treatment of RAW 264.7 cells, murine bone marrow-derived macrophages, and the human monocyte cell line THP-1 resulted in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A resulted in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibited this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial, as pretreatment of THP-1 cells with the monoclonal antibody 60b, which blocks this binding, inhibited JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction.

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.

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.