Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus.

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.

Interleukin 1 alpha activates two forms of p54 alpha mitogen-activated protein kinase in rabbit liver.

We have identified in rabbits two hepatic forms of T669 peptide kinases that are very strongly activated after systemic injection with the inflammatory cytokine interleukin 1 (IL-1). The T669 peptide contains a major phosphorylation site of the epidermal growth factor receptor, threonine 699 and is a substrate for mitogen-activated protein (MAP) kinases. The kinases were purified to homogeneity and corresponded to 50- and 55-kD proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequencing of 12 tryptic peptides of both kinases identified them as p54 MAP kinase alpha. This kinase belongs to the novel family of stress-activated protein kinases. This is the first evidence of IL-1 activating a specific protein kinase in vivo.

Association of mitogen-activated protein kinases with microtubules in mouse macrophages.

Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole-disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule-associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.

Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta.

Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta. These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A.

Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ([Ca2+]i) that parallels the activation of Ca2+-calmodulin-dependent protein kinase IV (CaMKIV), a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). However, inactivation of CaMKIV occurs despite the sustained increase in [Ca2+]i that is required for T cell activation. A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A (PP2A) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling. In Jurkat T cells, inhibition of PP2A activity by small t antigen enhanced activation of CREB-mediated transcription by CaMKIV. These findings reveal an intracellular signaling mechanism whereby a protein serine-threonine kinase (CaMKIV) is regulated by a tightly associated protein serine-threonine phosphatase (PP2A).

CaMKIIbeta association with the actin cytoskeleton is regulated by alternative splicing.

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII)beta has morphogenic functions in neurons not shared by the alpha isoform. CaMKIIbeta contains three exons (v1, v3, and v4) not present in the CaMKIIalpha gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIbeta, but not alpha, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIbeta association with the F-actin cytoskeleton within cells. CaMKIIbetae, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIbeta', which instead lacks exon v4, associated with F-actin as full-length CaMKIIbeta. Previous studies with CaMKIIbeta mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin-targeted CaMKIIbeta, but not alpha, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca(2+)/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIbeta' and betaM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIbeta variants also outside the nervous system.

Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals.

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase makes it Ca2+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. We show that autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase which requires two molecules of calmodulin. Ca2+/calmodulin acts not only to activate the "kinase" subunit but also to present effectively the "substrate" subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process that is inefficient at low occupancy of calmodulin. Simulations show that repetitive Ca2+ pulses at limiting calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the response of the kinase to sequential Ca2+ transients and establish a threshold frequency at which the enzyme becomes highly active.

Dual kinase-mediated regulation of PITK by CaMKII and GSK3.

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIdelta), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP.

Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses. We questioned whether these differences might reflect patterns of intracellular signal transduction. Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk. Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs). Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation. Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk. Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF. Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk. A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP. These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses.

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii.

BACKGROUND: Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. RESULTS: Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 - 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. CONCLUSION: This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi.

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis.

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.

Listeria monocytogenes, an invasive bacterium, stimulates MAP kinase upon attachment to epithelial cells.

Protein tyrosine phosphorylation is an important regulatory mechanism for many cellular processes in eucaryotic cells. During the invasion of the gram-positive pathogen, Listeria monocytogenes, into host epithelial cells, two host proteins become tyrosine phosphorylated. We have identified these major tyrosine phosphorylated species to be two isoforms of mitogen-activated protein (MAP) kinase, the 42 and 44 kDa MAP kinases. This activation begins within 5 to 15 min of bacterial infection. The tyrosine kinase inhibitor, genistein, blocks invasion as well as the tyrosine phosphorylation of these MAP kinases. Using cytochalasin D to block bacterial internalization but not adhesion, we showed that bacterial adherence rather than uptake is required for MAP kinase activation. Internalin mutants, which are unable to adhere efficiently to host cells, do not trigger MAP kinase activation. Other invasive bacteria, including enteropathogenic Escherichia coli (EPEC), and E. coli expressing Yersinia enterocolitica invasion, were not observed to activate MAP kinase during invasion into cultured epithelial cells. These results suggest that L. monocytogenes activates MAP kinase during invasion and a MAP kinase signal transduction pathway may be involved in mediating bacterial uptake.

Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2.

We recently showed that EGF and anisomycin activate two kinases, p45 and p55, whose distinguishing feature is that their detection in in-gel kinase assays is enhanced by copolymerised poly-Glu/Tyr or poly-Glu/Phe (Cano E, Hazzalin CA and Mahadevan LC, Mol. Cell. Biol., 20:117-121). Their activation characteristics and sizes are strikingly similar to those of JNK/SAPKs, which are also strongly activated by anisomycin. However, we show here that p45 and p55 are not JNK/SAPKs but murine forms of MAPKAP kinase-2 because: (i) Detection of immunoprecipitated JNK/SAPKs is completely dependent on the presence of c-Jun as substrate in the in-gel kinase assays, whereas detection of p45 and p55 is not. (ii) Detection of p45 and p55 activity is enhanced by the presence of poly-Glu/Tyr or poly-Glu/Phe, whereas JNK/SAPKs are not detectable under these conditions. (iii) Although the sizes of the murine JNK/SAPKs and MAPKAP K-2 are similar, human JNK/SAPKs migrate at 45 and 55 kDa whereas human MAPKAP K-2 migrates at 50 kDa; the poly-Glu/Tyr-enhanced activity in human cells migrates at 50 KDa. (iv) Purified rabbit muscle MAPKAP K-2 is detectable as two bands of activity on in-gel kinase assays and their detection is enhanced by poly-Glu/Tyr. (v) Finally, the anisomycin-activated poly-Glu/Tyr-enhanced p45 and p55 kinases can be immunoprecipitated from murine cells using an anti-MAPKAP K-2 antibody. Thus, EGF- and anisomycin-activated p45 and p55 are not JNK/SAPKs but MAPKAP K-2, implying that both these agents activate the p38/RK MAP kinase cascade.

Differential roles of Ca(2+)/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation.

The roles of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAPK) in long-term potentiation (LTP) were investigated in the CA1 area of hippocampal slices, using electrophysiological and biochemical approaches. A brief high-frequency stimulation, but not low-frequency stimulation, delivered to Schaffer collateral/commissural afferents produced a stable LTP and activated both CaM kinase II and 42 kDa MAPK. Different from the activity of CaM kinase II, the increase in MAPK activity was transient. At a concentration of 50 microM, but not of 30 microM, PD098059, a potent inhibitor of MAPK kinase, markedly inhibited the induction of LTP. Although the two concentrations had similar inhibitory effects on MAPK activity, only 50 microM PD098059 suppressed the activation of CaM kinase II. Application of calmidazolium, an antagonist of calmodulin, blocked both CaM kinase II activation and the LTP induction without affecting the increase in 42 kDa MAPK activity. Application of neurotrophin brain-derived neurotrophic factor (BDNF) promoted the induction of LTP, with concomitant activation of CaM kinase II. Under the same conditions, BDNF failed to activate MAPK in hippocampal slices. These results indicate that, although the LTP induction is accompanied by increases in two kinase activities, only CaM kinase II activation is required for this event.

Identification and purification of Ca2+/calmodulin-dependent protein kinase V from human gastric carcinoma.

We previously purified a novel Ca2+/calmodulin-dependent protein kinase (CaM kinase) V, which has proven to be a member of the CaM kinase I family. Immunohistochemical staining of surgically-resected specimens from human subjects using specific antibody which reacts with CaM kinases I and V demonstrated heterogeneous distribution of CaM kinase I/V in normal gastric mucosa. The kinase was located mainly at the bottom of foveoral epithelium and in the gastric gland (< 25% immunopositive). In contrast, this kinase was abundant in various types of gastric carcinomas (> 75%), but not in gastric adenomas. Preferential and consistent presence of this kinase was confirmed by immunoblot analysis of gastric carcinoma and human gastric cancer cell lines, Kato-III and MKN-45. CaM kinase I/V was co-purified with CaM kinase II from resected gastric carcinoma using anion-exchange chromatography followed by calmodulin-affinity chromatography. The two kinases were finally separated by HPLC-based gel filtration. Purified CaM kinase I/V from gastric carcinoma did not possess detectable autophosphorylating activity, in contrast to CaM kinase II. The findings suggest CaM kinase I/V may possess abnormal biochemical properties in human gastric carcinoma, and the kinase could participate in cell growth of the carcinoma.

Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms.

We have identified, expressed and characterized two new isoforms of the multifunctional Ca2+/calmodulin-dependent kinase (CaM kinase) cloned from rat heart. Both isoforms are variants of the neuronal delta-CaM kinase (termed delta A-CaM kinase), and are designated as delta B-CaM kinase and delta C-CaM kinase. The new isoforms differ from delta A-CaM kinase in its isoform-specific insert region, between nucleotides 984 to 1087 of the delta A-CaM kinase cDNA. Replacing these 102 nucleotides, a sequence of 33 nucleotides which code for 11 amino acids (KRKSSSSQMM) are introduced in delta B-CaM kinase. The delta C-CaM kinase lacks all 102 nucleotides and the corresponding 34 amino acids which they encode. The predicted molecular masses of the delta B- and delta C-CaM kinase isoforms are 57,697 Da and 56,446 Da, respectively. Recombinant delta-CaM kinases purified from transfected COS-7 cells were found to associate into a larger holoenzyme estimated to contain 8 to 10 subunits. The relative subunit molecular masses on SDS-polyacrylamide gel electrophoresis are 59 kDa, 54 kDa and 52 kDa for delta A-, delta B- and delta C-CaM kinase, respectively. All three isoforms showed a strict dependence on Ca2+/calmodulin for activity and exhibited the Ca(2+)-dependent autophosphorylation and resultant conversion to Ca(2+)-independent kinase activity, characteristic features of multifunctional CaM kinase. Phosphopeptide analysis after partial CNBr digestion suggests that delta B-CaM kinase is the predominant soluble CaM kinase species purified from rat heart.

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.

Skeletal muscle mitogen-activated protein kinases and ribosomal S6 kinases. Suppression in chronic diabetic rats and reversal by vanadium.

The mitogen-activated protein (MAP) kinases and ribosomal S6 protein kinases in the skeletal muscle of insulin-resistant long-term (2 and 6 months' duration) diabetic rats were investigated to understand further the changes in insulin intracellular signaling pathways that accompany diabetes. The effects of insulin-mimetic vanadium compounds on the activity of these kinases were also examined. In the insulin-resistant 2-month diabetic rats, the basal activities of MAP kinases were relatively unchanged, while the basal activities of S6 kinases were significantly increased. Intravenous injection of insulin moderately activated both the 42-kDa MAP kinase (p42mapk) and a 44-kDa MAP kinase (p44erk1) in the 2-month control rats but not in the 2-month diabetic rats. Insulin treatment markedly stimulated the activity of a novel 31-kDa S6 kinase and the previously described 90-kDa ribosomal S6 kinase encoded by one of the rsk genes (p90rsk) in the 2-month control rats, while the effect was substantially reduced in the diabetic rats. In the 6-month diabetic rats, the basal phosphotransferase activities of both MAP kinases were depressed threefold or greater. This correlated with reductions in the amount of immunoreactive p42mapk and p44erk1 proteins in extracts from the diabetic rats. The basal activity of the 31-kDa S6 kinase activity was also reduced fourfold in the 6-month diabetic rats. Treatment of the 2-month diabetic rats with vanadyl sulfate resulted in euglycemia, prevented the increase in the basal activity of S6 kinase, and improved the activation of S6 kinase by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag.

The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1). The cell constants are a = 49.32 A, b = 71.42 A, c = 61.25 A, and beta = 109.75 degrees, and the crystals diffract to better than 1.8 A resolution.

Phosphorylation of phospholamban in aortic smooth muscle cells and heart by calcium/calmodulin-dependent protein kinase II.

Phospholamban is a negative regulator of the sarcoplasmic reticulum Ca(2+)-pumping ATPase. Phosphorylation of phospholamban activates the ATPase and decreases the level of cytosolic calcium. Phospholamban is phosphorylated in heart by cAMP-dependent protein kinase, cGMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II (CM-kinase-II) and in smooth muscle cells by cGMP-dependent protein kinase. In contrast to heart muscle, phospholamban is poorly phosphorylated by CM-kinase-II in extracts of rat aortic smooth muscle cells. Rat aorta phospholamban amino acid sequence was identical to dog heart. The peptide substrate specificity of CM-kinase-II from rat aorta was the same as that from rat heart. The lack of phosphorylation of rat aorta phospholamban by the CM-kinase-II appears to result from the relatively low abundance of phospholamban in smooth muscle.