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PubMed1987

Phorbol ester- and light-induced endogenous phosphorylation of rat rod outer-segment proteins.

We have previously described the presence of a C-kinase in bovine retinal rod outer segments (ROS) (Kapoor and Chader, 1984). In this study, we have labeled rat retinas with freshly neutralized radiolabeled sodium phosphate (32P or 33P) by intravitreal injection and compared the phosphorylation patterns of ROS proteins induced by light and specific activators of the C-kinase phosphorylation system. Except for light treatment, all procedures were carried out in complete darkness using an infrared image converter. Incubation of 33P-labeled retinas in light for 5 min resulted in the phosphorylation of rhodopsin, 80-, 65-, 47-, 44-, and 15,000 MW proteins of crude ROS. Incubation of 33P-labeled retinas with 0.5 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the phosphorylation of several proteins including those at 80-, 65-, 47-, 44-, and 15,000 MW in crude ROS. ROS prepared in complete darkness did not exhibit any phosphorylation of proteins whereas ROS prepared in red light exhibited variable low phosphorylation of 80-, 47-, 44- and 15,000 MW proteins. 1-oleoyl-2-acetylglycerol (OAG) at 500 micrograms ml-1 caused the phosphorylation of the same proteins as observed with TPA. TPA (0.5-500 microM) and OAG (150-500 micrograms ml-1) did not induce rhodopsin phosphorylation. When purified ROS were prepared from 33P-pre-labeled retinas, the complete darkness control did not exhibit phosphorylation of any proteins. TPA, however, induced the phosphorylation of 80- and 65,000 MW proteins and light induced the phosphorylation of 80-, 65,000 MW proteins as well as opsin monomer and dimer. Affinity chromatography of phosphorylated ROS proteins on con A-Sepharose revealed that TPA does not induce rhodopsin phosphorylation whereas light does. Since light and TPA induced the phosphorylation of 80- and 65,000 MW proteins in ROS, it is possible to suggest at least a partial linkage of light- and C-kinase-mediated effects in situ.

Animals↗
PubMedDate not supplied

Intracellular calcium and alpha 1b-adrenoceptor phosphorylation.

BACKGROUND: Desensitization of G protein-coupled receptors is associated with receptor phosphorylation. Two groups of kinases seem to participate in such receptor phosphorylation, i.e., second messenger-activated protein kinases and G protein-coupled receptor kinases. Calcium seems to play a role in the phosphorylation of some G protein-coupled receptors. The role of calcium in alpha 1b-adrenoceptor phosphorylation has not been critically assessed. METHODS: Rat-1 fibroblasts stably expressing the hamster alpha 1b-adrenergic receptor were used. To study receptor phosphorylation cells metabolically labeled with [32P]Pi were lysed and the receptor immunoprecipitated using a polyclonal antibody generated against the receptor carboxyl terminal decapeptide. Intracellular calcium was determined by using Fura-2 fluorescence. RESULTS: Norepinephrine, endothelin-1, and lysophosphatidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce alpha 1b-adrenoceptor phosphorylation. The intracellular chelator, BAPTA, abolished the increase in intracellular calcium induced by the previously mentioned agents but did not affect the receptor phosphorylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slightly but consistently decreased. Thapsigargin increased intracellular calcium concentration but was unable to induce alpha 1b-adrenoceptor phosphorylation and decreased PMA-induced receptor phosphorylation. No increase in receptor phosphorylation was observed when calcium ionophores were used. CONCLUSIONS: Our data indicate that an increase in [Ca2+]i is not sufficient to induce alpha 1b-adrenoceptor phosphorylation and that buffering of [Ca2+]i does not alter the receptor phosphorylation induced by norepinephrine, lysophosphatidic acid, and PMA. A marginal role of calcium in the alpha 1b-adrenoceptor phosphorylation induced by endothelin-1 cannot be discarded.

Animals↗
PubMed1999

The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation.

BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.

Amino Acid Sequence↗
PubMed2002

Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins.

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.

Chromatography, High Pressure Liquid↗
PubMed1994

Calmodulin-activated phosphorylation of dystrophin.

Purified dystrophin glycoprotein complex (DGC) contains an endogenous protein kinase activity which phosphorylates dystrophin. Mg2+ (or Mn2+) and ATP are required for this phosphorylation. Ca(2+)-calmodulin increases the rate of phosphorylation of dystrophin 12-fold relative to the EGTA control, while other protein kinase activators, cAMP and cGMP, have no effect. Phosphorylation of other proteins in the DGC preparation was observed, with a 59-kDa protein also being phosphorylated in a calmodulin-dependent manner. These phosphorylations were all on serine residues. The DGC protein kinase activity also phosphorylates syntide-2, a peptide substrate for CaM kinase II, and antibodies raised against CaM kinase II cross-react with DGC blotted onto nitrocellulose. Further, purified, baculovirus-expressed CaM kinase II phosphorylates dystrophin and also phosphorylates at least one of the peptides of dystrophin which is phosphorylated by the DGC protein kinase activity, as shown by tryptic peptide maps. CaM kinase II also phosphorylates other proteins present in the DGC preparation that are phosphorylated by the endogenous protein kinase. Finally, dystrophin sequence 2618-3074, produced by recombinant techniques, is phosphorylated by both the DGC protein kinase and purified CaM kinase II. Since dystrophin and two other DGC components have also been shown to bind calmodulin, two important components of signal transduction--calmodulin binding and protein phosphorylation--operate in the DGC.

Animals↗
PubMed1998

Agonist-dependent phosphorylation of the parathyroid hormone/parathyroid hormone-related peptide receptor.

Native PTH/PTHrP receptors in ROS 17/2.8 cells are downregulated after PTH treatment. Since downregulation may involve receptor phosphorylation, we examined PTH/PTHrP receptor phosphorylation in ROS 17/2.8 cells and we mapped the agonist-induced phosphorylation sites using recombinant PTH/PTHrP receptors expressed in LLCPK-1 and COS-7 cells. The data show that the PTH/PTHrP receptor is rapidly phosphorylated in ROS 17/2.8 cells with a maximum occurring at 20 min. The phosphorylation was dose-dependent; it occurred with PTH concentrations that are known to downregulate the PTH/PTHrP receptor in ROS 17/2.8 cells. The time course and the dose requirement for phosphorylation were similar in ROS 17/2.8 cells, which express native PTH/PTHrP receptors, and in LLCPK-1 cells stably transfected with the PTH/PTHrP receptor cDNA. PTH/PTHrP receptor phosphorylation in ROS 17/2.8, COS-7, and LLCPK-1 cells was also stimulated with forskolin and phorbol myristate acetate (PMA). Additionally, in LLCPK-1 cells, which express native clacitonin receptors, PTH/PTHrP receptor phosphorylation was stimulated by calcitonin. These data suggest involvement of second messenger-stimulated kinases in PTH/PTHrP receptor phosphorylation. However, staurosporine, which fully blocked the effects of PMA, forskolin, and clacitonin, partially decreased the effects of PTH on PTH/PTHrP receptor phosphorylation. These data indicate involvement of other kinase(s) in PTH-induced PTH/PTHrP receptor phosphorylation. CNBr cleavage of recombinant receptors expressed in COS-7 cells combined with site-directed mutagenesis revealed that the phosphorylated residues of the PTH/PTHrP receptor map to two regions of the carboxyl-terminal tail located between residues A480 and M499 and residues M499 and M553. These data indicate that the PTH/PTHrP receptor is phosphorylated after PTH stimulation on two regions of the carboxyl-terminal tail and that agonist-dependent phosphorylation involves both staurosporine-sensitive and -insensitive kinases.

Amino Acid Sequence↗
PubMed1998

Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins.

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles.

KRP (telokin), an independently expressed C-terminal myosin-binding domain of smooth muscle myosin light chain kinase (MLCK), has been reported to have two related functions. First, KRP stabilizes myosin filaments (Shirinsky et al., 1993, J. Biol. Chem. 268, 16578-16583) in the presence of ATP. Secondly, KRP can modulate the level of myosin light chain phosphorylation. In this latter role, multiple mechanisms have been suggested. One hypothesis is that light chain phosphorylation is diminished by the direct competition of KRP and MLCK for myosin, resulting in a loss of contraction. Alternatively, KRP, through an unidentified mechanism, accelerates myosin light chain dephosphorylation in a manner possibly enhanced by KRP phosphorylation. Here, we demonstrate that KRP is a major phosphoprotein in smooth muscle, and use a comparative approach to investigate how its phosphorylation correlates with sustained contraction and forskolin-induced relaxation. Forskolin relaxation of precontracted artery strips caused little increase in KRP phosphorylation, while treatment with phorbol ester increased the level of KRP phosphorylation without a subsequent change in contractility. Although phorbol ester does not induce contraction of phasic tissues, the level of KRP phosphorylation is increased. Phosphopeptide maps of KRP from both tissues revealed multiple sites of phosphorylation within the N-terminal region of KRP. Phosphopeptide maps of KRP from gizzard were more complex than those for KRP from artery consistent with heterogeneity at the amino terminus and/or additional sites. We discovered through analysis of KRP phosphorylation in vitro that Ser12, Ser15 and Ser15 are phosphorylated by cAMP-dependent protein kinase, mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), respectively. Phosphorylation by GSK3 was dependent upon prephosphorylation by MAP kinase. This appears to be the first report of conditional or hierarchical phosphorylation of KRP. Peptides consistent with such multiple phosphorylations were found on the in vivo phosphopeptide maps of avian KRP. Collectively, the available data indicate that there is a complex relationship between the in vivo phosphorylation states of KRP and its effects on relaxation in smooth muscle.

Animals↗
PubMed2003

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain.

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

Adenosine Triphosphatases↗
PubMed1981

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.

Animals↗
PubMed1996

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Animals↗
PubMed1997

Phosphorylation of erythropoietin receptors in the endoplasmic reticulum by pervanadate-mediated inhibition of tyrosine phosphatases.

Erythropoietin (EPO) is the major hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Ligand binding to the erythropoietin receptor (EPO-R), a member of the cytokine receptor family, triggers Tyr phosphorylation of the surface form of the receptor, presumably mediated by the Janus kinase (JAK) 2. To study whether non-surface EPO-R can be phosphorylated, Ba/F3 cells stably transfected with EPO-R were treated with pervanadate (PV), which is widely used as a potent tool to inhibit cellular protein Tyr phosphatases, thus resulting in enhanced Tyr phosphorylation of cellular proteins. PV treatment caused the EPO-R to undergo Tyr phosphorylation in a time-dependent and dose-dependent manner. PV-mediated Tyr phosphorylation of EPO-R occurred at several intracellular sites including the endoplasmic reticulum (ER), because both endoglycosidase H (endo H)-resistant EPO-R and the ER-retained EPO-R mutant (DeltaWS1 EPO-R) were Tyr phosphorylated in response to PV. Moreover, in metabolic labelling experiments, endo H-sensitive EPO-R was also phosphorylated. The phosphorylated fraction accounted for only 30-50% of the newly synthesized EPO-R, the fraction that normally exits from the ER. Tyr phosphorylation could not be detected on proteolytic fragments of the EPO-R, suggesting that this is a highly regulated process. Unlike the wild-type (wt) EPO-R, which was phosphorylated both on EPO binding and after inhibition of Tyr phosphatases by PV treatment, an EPO-R mutant (W282R EPO-R) that does not activate JAK2 was phosphorylated after PV treatment but not by EPO binding. Both EPO-R and JAK2 were phosphorylated with similar kinetics by PV treatment, suggesting that JAK2, as well as protein Tyr kinases different from JAK2, might mediate PV-dependent EPO-R phosphorylation. Furthermore the Tyr-phosphorylated ER-retained EPO-R mutant DeltaWS1 co-immunoprecipitated with JAK2 kinase, indicating that the EPO-R might interact with JAK2 while in the ER.

Animals↗
PubMed1999

Syk and paxillin are differentially phosphorylated following adhesion to the plastic substrate in rat alveolar macrophages.

Adhesion is associated with tyrosine phosphorylation in many types of cells. Although macrophages are known to adhere and phagocytose foreign particles, the signal transduction pathway of macrophages in response to adhesion to the foreign substrate has not been fully investigated. In the present study we investigated tyrosine-phosphorylated proteins and phosphorylation of paxillin in alveolar macrophages (AMs) following adhesion to a plastic substrate. Adhesion to a plastic dish resulted in tyrosine phosphorylation of a 68 000 MW protein, which was shown, by immunoprecipitation and immunoblotting in the present study, to be a rat Syk kinase. Treatment with erbstatin reduced both tyrosine phosphorylation of Syk and adherence of AMs, while treatment with cytochalasin B inhibited spreading of AMs but did not inhibit tyrosine phosphorylation of Syk. These results suggest that tyrosine phosphorylation of Syk plays an important role in adhesion of AMs to the plastic substrate, but not in AM spreading. Paxillin is known to be tyrosine phosphorylated following adhesion to the extracellular matrix in many types of cells. However, paxillin appeared to be serine/threonine phosphorylated rather than tyrosine phosphorylated following adhesion of AMs to the plastic substrate. Treatment with A23187 (a calcium ionophore), but not phorbol 12-myristate 13-acetate (PMA; a protein kinase C stimulator), induced tyrosine phosphorylation of Syk in non-adherent AMs. Treatment with either A23187 or PMA caused electromobility changes of paxillin that were mainly a result of serine/threonine phosphorylation. These results suggest that adhesion to the plastic substrate leads to two differently regulated events in AMs: tyrosine phosphorylation of Syk and serine/threonine phosphorylation of paxillin, both of which are probably mediated by an increase in intracellular calcium.

Animals↗
PubMed1995

Guanosine 5'-3-O-(thio)triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells. Role of p21rho.

Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin. GTP gamma S induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 microM) and reached maximum levels after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M(r) 70,000-80,000 paxillin band. Guanosine 5'-O-(2-thiodiphosphate) inhibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 100 microM. Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation. Varying the Ca2+ concentration from 0 to 6 microM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTP gamma S to induce tyrosine phosphorylation. GTP gamma S was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+. Furthermore, 30 microM AlF4- only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTP gamma S to stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands including p125FAK and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21rho (p21rho17-44) inhibited GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 microM). This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin. In contrast, 20 microM p21ras17-44 peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTP gamma S stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.

3T3 Cells↗
PubMed1997

Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism.

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.

Adenosine Diphosphate↗
PubMed2004

Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status.

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser2448 as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser2448 phosphorylation via protein kinase B (PKB), while Ser2448 phosphorylation is attenuated with amino acid starvation. Here we have identified Thr2446 as a novel nutrient-regulated phosphorylation site on mTOR. Thr2446 becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr2446 and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr2446 is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser2448 is phosphorylated. These data suggest differential phosphorylation Thr2446 and Ser2448 could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.

AMP-Activated Protein Kinases↗
PubMed2000

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine.

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.

Amino Acid Substitution↗