Phosphorylation of rat liver inorganic pyrophosphatase by ATP in the absence and in the presence of protein kinase

Cytoplasmic inorganic pyrophosphatase of rat liver can be phosphorylated by cAMP-dependent protein kinase on a serine residue with a concomitant increase in enzymic activity. Phosphorylation is also observed in the absence of protein kinase, but in this case much higher concentrations of ATP are required and the stability characteristics of the phosphoenzyme resemble those of an acyl phosphate. Kinase-free phosphorylation of the animal inorganic pyrophosphatase, unlike that of microbial pyrophosphatases, does not activate the enzyme. Pyrophosphatase may thus provide a new example of an enzyme whose evolution involves convergence of regulatory phosphorylation mechanisms.     

Phosphorylation of intracellular precursors of human IL-1

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.     

Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.     

Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain.

The in vitro phosphorylation of chicken desmin by the catalytic subunit of cAMP-dependent protein kinase was analysed. Phosphorylated desmin loses the ability to form intermediate filaments (IFs). Fragmentation at the sole cysteine and mild chymotryptic treatment show a differential phosphorylation of the three structural domains. Only the amino-terminal head domain is the target of the kinase. Peptide analysis shows that serine 29 is fully phosphorylated, while serine 35 and 50 are phosphorylated at least at 22 and 50% respectively. All three sites show the sequence arginine-X-serine with X being a small residue. These results strengthen the view that the nonhelical head domain has a strong influence on filament integrity most likely via a direct influence of some of its arginine residues. Taken together with previous results (Inagaki et al., 1987) on the phosphorylation of vimentin by kinase A, a new view on IFs emerges. Phosphorylation could allow for regulatory processes in assembly and turnover.     

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.     

Analysis of a bacterial lipopolysaccharide-activated serine kinase that phosphorylates p65/L-plastin in macrophages

We previously identified p65/L-plastin as a phosphorylated protein in LPS-stimulated macrophages and determined its phosphorylation site. In vitro kinase assay using peptide substrates revealed that LPS-stimulated kinase activity selectively phosphorylated their serine-5 (Ser-5) residue. Kinase inhibitors for cAMP-dependent kinase such as H-89 inhibited the Ser-5 phosphorylation, but cAMP was not essential for the kinase activity. The LPS-stimulated kinase activity in cytosol fractions of macrophages was recovered as a sharp peak by anion exchange chromatography. These findings suggest that an as yet unknown H-89-sensitive serine kinase is rapidly activated by LPS stimulation and then phosphorylates p65/L-plastin, playing a vital role in macrophage activation.     

Phosphorylase kinase specificity. A comparative study with cAMP-dependent protein kinase on synthetic peptides and peptide analogs of glycogen synthase and phosphorylase

A synthetic pentadecapeptide, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu, corresponding to the phosphorylatable site at the NH2 terminus of glycogen synthase, could be phosphorylated stoichiometrically at seryl residue 7 by both phosphorylase kinase and cAMP-dependent protein kinase. Phosphorylation of seryl residue 3 also occurred after prolonged incubation with cAMP-dependent protein kinase. Kinetic studies show that the pentadecapeptide is a better substrate for phosphorylase kinase. A peptide consisting of residues 1-11 was not as good a substrate and substitution of Arg-4 by Lys and Ser-9 by ARg in the unidecapeptide decreased and increased phosphorylase kinase reaction rates, respectively. Higher rates of phosphorylation were obtained with peptides of the phosphorylatable site of phosphorylase. A peptide with the sequence, Leu-Ser-Tyr-Arg-Arg-Tyr-Ser-Leu was phosphorylated initially by phosphorylase kinase and cAMP-dependent protein kinase at Ser-2 and Ser-7, respectively. Upon longer incubation, second site phosphorylation occurred with both kinases. A peptide of the same sequence with D-amino acids could not be phosphorylated but was a competitive inhibitor of both enzymes. The results suggest that optimal interaction of the two kinases depends on various factors including the orientation of arginyl groups with respect to the phosphorylatable serine.     

Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase

The phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase I is markedly enhanced if the enzyme has previously been phosphorylated by cAMP-dependent protein kinase. The presence of phosphate in the primary cAMP-dependent protein kinase sites, sites 1a, 1b, and 2 (serine 7), increases the activity of casein kinase I toward residues in the vicinity of these sites. This synergistic phosphorylation correlates with potent inactivation of the glycogen synthase. Analysis of the NH2 terminus of the enzyme subunit indicated that phosphorylation at serine 7 caused serine 10 to become a preferred casein kinase I site and that phosphoserine can be an important recognition determinant for casein kinase I. This finding can also explain how epinephrine stimulation of skeletal muscle provokes significant increases in the phosphorylation state of serine residues, in particular serine 10, not recognized by cAMP-dependent protein kinase.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.