Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Houssaye, Becky A. de la; Masaracchia, Ruthann A.

doi:10.1016/0003-2697(83)90343-3

Cited by 17 publications

(13 citation statements)

References 12 publications

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“…5A) can be distinguished from PKC (77000) [65], PAK 11 (80000) [35], histone-4 PK (68000) [36] and casein kinase I (36000) [63], values referring to rat brain, rabbit reticulocyte, murine sarcoma and murine 3T3 L1 cells, respectively. Third, as shown in Fig.…”

Section: Eurly-eluting S6-phosphoryluting Activitymentioning

confidence: 99%

Mitogen‐responsive S6 kinase

Lawen

Burger

Martini

1989

European Journal of Biochemistry

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Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin ~ cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K + concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent M , of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56000 (determination in the presence of 200 mM KCI in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase 11, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogenresponsive S6 kinase(s) [Martini, 0. H. W. and Lawen, A. (1985) [14, 351, and fertilization of oocytes [16]. Serum-induced S6 phosphorylation was promptly reversed when cells were exposed to stress, a condition that also causes inhibition of translation [17].As an end-point of signalling pathways, S6 can be utilized, in analogy to phosphorylase [IS], to unravel signal flux by proceeding from distal to proximal. Towards this goal we have sought the S6-phosphorylating kinase(s) mediating growth- Abbreviations. PKC, CaZ +-phosphatidylserine-diolein-dependent protein kinase (protein kinase C); CAMP-PK, CAMP-dependent protein kinase; PAK 11, protease-activated protein kinase 11; histone-4 PK, histonc-4 phosphotransferase; S6-KI1, Xenopus oocyte S6 kinase I1 (two closely related proteins referred to as SG-KIIa and S6-KIIP); NaCI/Pi, phosphate-buffered salinc.Enzyme. Protein kinases (EC 2.7.1.37). promoting signals. To obviate possible interference by CAMPdependent protein kinase (CAMP-PK) (the enzyme has been implicated in both negative, antagonizing anabolic signals [19], and positive [20 -241 control of S6 phosphorylation), the search was begun in S49 kin-murine lymphoma cells which express CAMP-PK at a subnormal level [25]. A strong S6-phosphorylating activity was found, which was effector-independent and chromatographically distinct from several effector-dependent Sh-phosphorylating kinases of broader substrate range. A similar human enzyme(s) was then detected using HeLa S3 cells, extending the pioneering work of McConkey an...

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Section: Eurly-eluting S6-phosphoryluting Activitymentioning

confidence: 99%

Mitogen‐responsive S6 kinase

Lawen

Burger

Martini

1989

European Journal of Biochemistry

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“…The protease-activated H4 protein kinase has been shown to phosphorylate a synthetic peptide with the sequence ValLys-Arg-Ile-Ser-Gly-Leu [14,15]. This sequence corresponds to residues 43 -49 in the histone H4 [17,18].…”

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confidence: 99%

“…The H4 phosphotransferase is clearly not a cyclic-nucleotide-dependent enzyme [14,16], although the regulation of the enzyme in vivo has not been resolved [15]. However, the occurrence of basic residues amino-terminal to the modified serine suggests that the substrate specificity may be similar to the cyclic-AMP-dependent or cyclic-GMP-dependent enzyme [8,12].…”

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confidence: 99%

Primary Substrate Specificity Determinants for the H4‐Specific Protease‐Activated Protein Phosphotransferase

Eckols

Thompson

Masaracchia

1983

European Journal of Biochemistry

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The specificity of the histone-H4-specific, protease-activated protein kinase (H4-PK) was examined using two series of synthetic peptides corresponding to the phosphorylation sites in histone H4 and pyruvate kinase. Optimum kinetic constants for phosphorylation were observed using the peptide Val-Lys-Arg-Ile-Ser-Gly-Leu. Peptides in which the Lys was replaced by Arg or the Lys-Arg sequence was transposed were phosphorylated with less favorable kinetics. Peptides with either basic residue deleted did not serve as substrates. Only the H4 peptide, containing an Arg-Arg sequence, was phosphorylated by the cyclic-AMP-dependent protein kinase (CA-PK). Distinct specificity determinants for H4-PK and CA-PK were also observed using the pyruvate kinase peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly). Collectively the data indicated that the primary substrate specificity determinants for H4-PK are Lys-Arg-Xaa-Ser whereas the CA-PK selectively phosphorylates the sequence Arg-Arg-Xaa-Ser.Since the observation that glycogen phosphorylase activity is regulated by covalent phosphorylation catalyzed by phosphorylase kinase [I], the identification of distinct protein phosphotransferases (protein kinases) and the association of these enzymes with unique protein substrates has received widespread attention [2 -41. Initial studies proposed that the specificity determinants which defined a phosphorylation site were determined by higher orders of protein structure [4,5]. However, the observations that denaturation increased the ability of some proteins to serve as protein kinase substrates [6,7] and that small synthetic peptides could be phosphorylated in vitro [8 -121 have indicated that the substrate specificity determinants were dictated, at least in part, by the primary sequence.The observed kinetic constants, determined in experiments utilizing the cyclic-AMP-dependent protein kinase catalytic subunit and synthetic peptides derived from the primary sequence at the phosphorylation site in pyruvate kinase (Leu-Arg-Arg-Ser-Leu-Gly), indicated that multiple basic residues were required in the proximity of, and aminoterminal to, the modified serine [9]. Additional studies [lo, 1 I], utilizing synthetic peptides with further modification in the pyruvate kinase sequence, suggested that the cyclic-AMPdependent protein kinase selectively phosphorylated serine residues in a sequence of the type Arg-Arg-Xaa-Ser or LysArg-Xaa-Yaa-Ser. The positive correlation between the specificity determinants predicted in the peptide studies and the primary sequence at phosphorylation sites in several physiological protein substrates has provided compelling evidence that the primary sequence substantially determines the substrate specificity of the cyclic-AMP-dependent protein kinase [13].

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“…Because mutants lacking cAMP production or cAMP-dependent protein kinase activity under nonpermissive conditions cannot grow (24; unpublished data), the gual strain must contain this enzyme. Since the purified cAMP-dependent protein kinases from yeasts and the pure catalytic subunit from mammalian tissue do not require guanine nucleotides for their activity (12,30,36), it is unlikely that this enzyme is affected by guanine deprivation.…”

Section: Discussionmentioning

confidence: 99%

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Olempska-Beer¹,

Freese²

1987

Mol. Cell. Biol.

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Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, (i) cAMP deficiency is not required for initiation of meiosis and sporulation, (ii) cAMP is produced in excess and excreted to the medium, (iii) the cells sporulate better if the cAMP concentration is increased by addition of IBMX, (iv) the cells require a gluconeogenic carbon source for complete and efficient sporulation, and (v) suppression of sporulation by glucose is not mediated by cAMP.In the laboratory, sporulation of Saccharomyces cerevisiae is usually initiated by nitrogen starvation of cells adapted to grow on gluconeogenic carbon sources (for reviews, see references 10 and 14). Our laboratory has shown that S. cerevisiae also sporulates in media rich in nitrogen in which only carbon or sulfur are limited (2, 3). Under these conditions, as well as under nitrogen starvation, guanine nucleotides (GTP and GDP) decrease while changes of other nucleoside tri-and diphosphates show no correlation with sporulation. Sporulation can also be initiated specifically by guanine deprivation of a guanine auxotroph (3, 16). These results show that sporulation can be initiated by alternative ways of starvation and suggest that the decrease of guanine nucleotides provides a specific sporulation signal.Matsumoto and his colleagues isolated and characterized mutants deficient in adenylate cyclase (cyri) or cAMPdependent protein kinase (CYR3) (22,24,36). They found that temperature-sensitive diploids homozygous for these mutations sporulated (at a low frequency) in a medium rich in nitrogen and containing acetate as the sole carbon source; the parent strain did not sporulate under these conditions. Therefore, they proposed that initiation of meiosis may require a low level of cAMP or absence of cAMP-dependent protein kinase (21, 23).We examined this proposal by studying the effects of guanine starvation on intracellular cAMP and sporulation in a guanine auxotroph. We compared the results of four experiments in which sta...

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Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Cited by 17 publications

References 12 publications

Mitogen‐responsive S6 kinase

Mitogen‐responsive S6 kinase

Primary Substrate Specificity Determinants for the H4‐Specific Protease‐Activated Protein Phosphotransferase

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

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