Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.     

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.     

Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites

A novel peptide with multiple phosphorylation sites, which we designated as multide, was developed to detect a wide variety of protein kinases in crude cell extracts. Multide, KKRKSSLRRWSPLTPRQMSFDC, has been designed to contain consensus sequences for various Ser/Thr protein kinases including cAMP-dependent protein kinase, protein kinase C, MAP kinases, and Ca(2+)/calmodulin-dependent protein kinases in a single peptide. In-gel protein kinase assay using multide was found to be very useful for analyzing the activities of protein kinases that are altered in response to various extracellular stimuli. The substrate specificities of the protein kinases thus detected were further determined by using five multide analogs with different phosphorylation sites.     

Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.     

Profiling substrate phosphorylation at the phosphopeptide level

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.     

Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.     

Identification of mitotic (CDC2) and interphase histone H1 kinases by nondenaturing gel electrophoresis and peptide assays

A new method of nondenaturing gel electrophoresis and a specific peptide based assay were used to study the histone kinases in mitotic and interphase mouse fibroblasts. The gels resolved four activities, one of which was shown to be the mitotic (CDC2) H1 kinase by virtue of its antigenicity. A new peptide substrate for the CDC2 kinase was phosphorylated by both S-phase and mitotic cell extracts and reacted with two protein kinases in the gels. Since the interphase enzyme did not react with the antibody, the results suggest that it is either a "masked" form of CDC2 or a second enzyme, functionally related to CDC2, which is responsible for the interphase phosphorylation of H1.     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Expression and purification of a convenient Ca2+-calmodulin-dependent protein kinase II GST-fusion substrate

Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.     

Expression and purification of a convenient Ca-calmodulin-dependent protein kinase II GST-fusion substrate

Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.     

A microtiter-based assay for the detection of protein tyrosine kinase activity

A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [³²P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Characterization of the insulin receptor kinase purified from human placental membranes

The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.     

A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here, we demonstrate that the phytochromes functionally interact with the catalytic subunit of a Ser/Thr-specific protein phosphatase 2A designated FyPP. The interactions were influenced by phosphorylation status and spectral conformation of the phytochromes. Recombinant FyPP efficiently dephosphorylated oat phytochrome A in the presence of Fe(2+) or Zn(2+) in a spectral form-dependent manner. FyPP was expressed predominantly in floral organs. Transgenic Arabidopsis plants with overexpressed or suppressed FyPP levels exhibited delayed or accelerated flowering, respectively, indicating that FyPP modulates phytochrome-mediated light signals in the timing of flowering. Accordingly, expression patterns of the clock genes in the long-day flowering pathway were altered greatly. These results indicate that a self-regulatory phytochrome kinase-phosphatase coupling is a key signaling component in the photoperiodic control of flowering.     

Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.

The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.     

Resistance of initiation factor 2 (eIF-2alpha) kinases to staurosporine: an approach for assaying the kinases in crude extracts.

We studied the effect of staurosporine on two well characterised mammalian eIF-2alpha kinases, the heme-regulated translational inhibitor (HRI), and interferon-inducible double-stranded RNA-activated protein kinase (PKR). Both pure eIF-2 and a synthetic peptide used to measure the activity of purified or immunoprecipitated enzymes (sequence ILLSELSRRRIRAI) were phosphorylated with purified enzymes and crude preparations of tissues or cells in the presence of the inhibitor. In the presence of 0.25 microM staurosporine (a concentration which completely inhibits a wide range of Ser/Thr protein kinases), the phosphorylation of eIF-2alpha by HRI and PKR was not inhibited. The lack of response of eIF-2alpha kinases to staurosporine allowed us to measure PKR activity in salt washed postmicrosomal supernatants without previous purification of the enzyme. In the presence of poly(I):poly(C), the PKR activator, we detected both an increased phosphorylation of eIF-2alpha and an increment in the autophosphorylation of PKR. We also confirmed an induction of PKR in cultured neuronal cells after treatment with interferon. The results obtained following phosphorylation of the synthetic peptide with crude extracts are less conclusive. Although its phosphorylation is specific because it displaces eIF-2 phosphorylation, and the presence of staurosporine prevents its phosphorylation by other serine/threonine kinases, it is a rather poor substrate for PKR.     

Different effects on activity caused by phosphorylation of tyrosine hydroxylase at serine 40 by three multifunctional protein kinases.

Tyrosine hydroxylase was maximally phosphorylated by protein kinase C, with a stoichiometry of 0.43 mol of phosphate/mol of tyrosine hydroxylase subunit at Ser40, and by calmodulin-dependent protein kinase II, with stoichiometries of 0.43 mol/mol at Ser40 and 0.76 mol/mol at Ser19, respectively, without undergoing any significant direct activation. In contrast, the enzyme was maximally phosphorylated with a stoichiometry of 0.78 mol of phosphate/mol of subunit at Ser40 by cAMP-dependent protein kinase, which resulted in a large activation of the enzyme (about 3-fold activation under the assay conditions). Incubation of the enzyme, which had previously been maximally phosphorylated by calmodulin-dependent protein kinase II, with protein kinase C under phosphorylating conditions resulted in no additional incorporation of phosphate into the enzyme, suggesting that both protein kinases phosphorylated Ser40 of the same subunits of the enzyme. Since tyrosine hydroxylase is thought to be composed of four identical subunits, the results may indicate that calmodulin-dependent protein kinase II or protein kinase C phosphorylates only two of the four subunits of the enzyme at Ser40 without affecting the enzyme activity and that cAMP-dependent protein kinase phosphorylates Ser40 of all four subunits of the enzyme molecule, causing a marked activation. Based on a linear relationship between phosphorylation and the resulting activation of the enzyme by cAMP-dependent protein kinase, possible mechanisms for the activation of the enzyme by the protein kinase are discussed.