Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.     

Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue.

The site of phosphorylation of phenylalanine ammonia-lyase (PAL) has been identified as a threonine residue. A Ca(2+)-stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease in Vmax in agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545 in the sequence VAKRTLTT (539-546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants.     

Calcium/calmodulin up-regulates a cytoplasmic receptor-like kinase in plants

Calcium/calmodulin-dependent kinases play an important role in protein phosphorylation in eukaryotes. However, not much is known about calcium/calmodulin-dependent protein phosphorylation and its role in signal transduction in plants. By using a protein-protein interaction-based approach, we have isolated a novel plant-specific calmodulin-binding receptor-like cytoplasmic kinase (CRCK1) from Arabidopsis thaliana, as well as its ortholog from Medicago sativa (alfalfa). CRCK1 does not show high homology to calcium/calmodulin-dependent protein kinases in animals. In contrast, it shows high homology in the kinase domain to serine/threonine receptor-like kinases in plants. However, it contains neither a transmembrane domain nor an extracellular domain. Calmodulin binds to CRCK1 in a calcium-dependent manner with an affinity of approximately 20.5 nm. The calmodulin-binding site in CRCK1 is located in amino acids 160-183, which overlap subdomain II of the kinase domain. CRCK1 undergoes autophosphorylation in the presence of Mg2+ at the threonine residue(s). The Km and Vmax values of CRCK1 for ATP are 1 microm and 33.6 pmol/mg/min, respectively. Calcium/calmodulin stimulates the kinase activity of CRCK1, which increases the Vmax of CRCK1 approximately 9-fold. The expression of CRCK1 is increased in response to stresses such as cold and salt and stress molecules such as abscisic acid and hydrogen peroxide. These results indicate the presence of a calcium/calmodulin-regulated receptor-like cytoplasmic kinase in plants. Furthermore, these results also suggest that calcium/calmodulin-regulated protein phosphorylation involving CRCK1 plays a role in stress signal transduction in plants.     

Transient increases in intracellular calcium result in prolonged site-selective increases in Tau phosphorylation through a glycogen synthase kinase 3beta-dependent pathway.

Calcium is a universal intracellular signaling molecule. Through variations in both the amplitude and frequency of intracellular calcium increases, the same calcium ion can elicit different responses. In this report, we investigated the effect of a calcium transient, lasting 2-5 min, on alterations in the phosphorylation state of the cytoskeletal protein, tau. Transient increases in calcium result in a prolonged (1-4 h) approximately 60% increase in tau phosphorylation at the Tau-1 epitope. These increases in tau phosphorylation appear to be more dependent upon the duration of the increase in intracellular calcium and less on the amplitude. The calcium-induced increases in tau phosphorylation are not dependent upon protein synthesis, nor are protein kinase C or calcium/calmodulin-dependent protein kinase II involved in the response. However, the calcium-induced increase in tau phosphorylation was inhibited by lithium, a noncompetitive inhibitor of glycogen synthase kinase-3beta (GSK-3beta), and by the tyrosine kinase inhibitor, genistein. Furthermore, transient increases in calcium resulted in a prolonged increase in GSK-3beta tyrosine phosphorylation concomitant with the increase in tau phosphorylation. Therefore, this study is the first to indicate that transient increases in intracellular calcium result in increased tyrosine phosphorylation and activation of GSK-3beta which subsequently results in a sustained increase in the phosphorylation state of tau.     

The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD1

The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

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.     

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3',5'-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Calpha independently and to the same extent as serine 67, the previous protein kinase Calpha-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.     

Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature

The present study aimed to determine the thermal response of the Mediterranean mussel Mytilus galloprovincialis by integrating information from various levels of biological organization including behavior, metabolic adjustments, heat shock protein expression, and protein kinase activity. Behavioral responses were determined by examining the effect of warming on valve closure and opening. Metabolic impacts were assessed by examining the activity of the key glycolytic enzyme pyruvate kinase (PK). Molecular responses were addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases, p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs). Mussels increased the duration of valve closure by about sixfold when acclimated to 24 degrees C rather than to 17 degrees C. As indicated by the activity of PK, such behavior caused metabolic depression and probably a shift from aerobic to anaerobic metabolism. Acclimation to temperatures higher than 24 degrees C caused an increase in mortality and induced the expression of Hsp72. Increased phosphorylation of p38 MAPK and JNKs indicated activation of MAPK signaling cascades. The potential involvement of MAPKs in the induction of Hsp genes in the tissues of M. galloprovincialis is discussed. In conclusion, it seems that M. galloprovincialis lives close to its acclimation limits and incipient lethal temperature and that a small degree of warming will elicit stress responses at whole organism and molecular levels.     

Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli

Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during Infection of Its host, Escherichia coli. The protein kinase (gpO.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphoryiated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electro-phoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which Is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-rnfected cells.     

Inhibition of yeast ribosomal stalk phosphorylation by Cu-Zn superoxide dismutase

Reversible phosphorylation of acidic ribosomal proteins of Saccharomyces cerevisiae is an important mechanism, regulating the number of active ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. A new protein-inhibitor regulating activity of PK60S kinase has been purified from yeast extracts and characterised. Peptide mass fingerprinting (PMF) and amino-acid sequence analysis by Post Source Decay (PSD) have identified the inhibitor as a Cu-Zn superoxide dismutase (SOD). Inhibition by SOD is competitive with respect to protein substrates-P proteins and 80S ribosome-with K(i) values of 3.7 microM for P2A protein and 0.6 microM for 80S ribosomes. A close correlation was found between the state of phosphorylation of P proteins in diauxic shift and logarithmic growth yeast cells and activity of SOD. The possible mechanism of regulation of PK60S activity, and participation of SOD protein in regulation of 80S-ribosome activity in stress conditions, is discussed.     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells

The effects of thrombin and histamine on protein phosphorylation in intact cultured human umbilical vein endothelial cells (HUVEC) prelabeled with 32PO4 were investigated. Incubation of HUVEC with either thrombin or histamine, agonists known to induce rapid transient increases in intracellular calcium levels in HUVEC, caused a rapid reversible increase in the phosphorylation of a protein with a Mr = 100,000 independent of the presence of extracellular calcium. Immunological and biochemical studies demonstrated that this Mr = 100,000 protein is elongation factor 2 (EF-2), a substrate previously shown to be phosphorylated by calcium/calmodulin-dependent protein kinase III (Nairn, A. C., and Palfrey, H. C. (1987) J. Biol. Chem. 262, 17299-17303). EF-2 is crucial for protein synthesis because it catalyzes the translocation of peptidyl-tRNA on the ribosome. Phosphoamino acid analysis of the EF-2 immunoprecipitated from HUVEC revealed that all of the thrombin-stimulated phosphorylation occurred on threonine. EF-2 was also phosphorylated when HUVEC were treated with the calcium ionophore, ionomycin. Phosphorylation of EF-2 was not increased by treatment with D-Phe-Pro-Arg-chloromethyl ketone thrombin, phorbol dibutyrate, forskolin, or 8-bromo-cGMP. The transient nature of the phosphorylation of EF-2 is consistent with it having a role in mediating some of the transient effects of thrombin and histamine on endothelial cell protein synthesis and functional capabilities.     

Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.     

Oxidant stress stimulates phosphorylation of eIF4E without an effect on global protein synthesis in smooth muscle cells. Lack of evidence for a role of H202 in angiotensin II-induced hypertrophy

Reactive oxygen species (ROS) are implicated in the pathogenesis of several proliferative diseases, including atherosclerosis and cancer. Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in cell proliferation and differentiation. To gain insight into molecular mechanisms by which ROS influence the pathogenesis of these diseases, I have studied the effect of H(2)O(2), a ROS, on eIF4E phosphorylation. H(2)O(2) induced eIF4E phosphorylation in a dose- and time-dependent manner in growth-arrested smooth muscle cells (SMC). H(2)O(2)-induced eIF4E phosphorylation occurred on serine residues. PD098059, a specific inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase inhibited ERK activities but had no significant effect on eIF4E phosphorylation induced by H(2)O(2). Similarly, SB203580, a specific inhibitor of p38 MAPK, although inhibiting H(2)O(2)-induced p38 MAPK activity, had no effect on H(2)O(2)-induced eIF4E phosphorylation. Calphostin C, a specific inhibitor of protein kinase C, also had no effect on H(2)O(2)-induced eIF4E phosphorylation. In contrast, trifluoperazine, an antagonist of calcium/calmodulin kinases, completely blocked H(2)O(2)-induced eIF4E phosphorylation. In addition, intracellular and extracellular Ca(2+) chelators significantly inhibited H(2)O(2)-induced eIF4E phosphorylation. Despite its ability to induce eIF4E phosphorylation, H(2)O(2) had no significant effect on protein levels and new protein synthesis as compared with control. In contrast, it induced the expression of c-Fos, c-Jun, and HSP70 in a time-dependent manner in SMC. Together, these results suggest that H(2)O(2), a ROS and a cellular oxidant, induces eIF4E phosphorylation in a manner that is dependent on Ca(2+) and Ca(2+)/calmodulin kinases and independent of ERKs, p38 MAPK, and protein kinase C. These results also suggest that enhanced eIF4E phosphorylation by H(2)O(2) appears to be an important event in SMC in response to oxidant stress and that eIF4E phosphorylation may be associated with the translation of a small subset of mRNAs such as c-fos, c-jun, and HSP70 gene mRNAs, whose products may have a critical role in cell survival.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.