Mechanism of Activation of cAMP-Dependent Protein Kinase: In Mucor rouxii the Apparent Specific Activity of the cAMP-Activated Holoenzyme Is Different than That of Its Free Catalytic Subunit

Zaremberg, Vanina; Donella‐Deana, Arianna; Moreno, Sílvia

doi:10.1006/abbi.2000.1948

Cited by 12 publications

(14 citation statements)

References 31 publications

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“…The authors also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms (27,28). We have shown, some years ago, that peptide substrates were involved in the activation of a fungal PKA by cAMP (29).…”

Section: Resultsmentioning

confidence: 89%

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Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

Galello

Portela

Moreno

et al. 2010

Journal of Biological Chemistry

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The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s ؊1 . We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P؉1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P؉1 is not critical. The best substrate has in position P؉4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates.Protein phosphorylation is one of the most fundamental mechanisms for signal transduction. The specificity of signal transduction depends upon the ability of each kinase to precisely phosphorylate particular sites on specific substrate proteins. Protein kinase A (PKA) 3 is one of the best characterized members of this protein family (1). Phosphorylation of its substrate targets is known to be critical for regulating a multitude of cellular processes, including metabolism, gene transcription, ion flux, growth, and cell death (2). The specificity in phosphorylation is determined by at least two major factors: peptide specificity and substrate and kinase localization. The effectiveness of protein phosphorylation by protein kinases in general, including PKA, is believed to depend on the primary structure of the protein around the phosphorylation site; synthetic peptides have thus been used to define the consensus phosphorylation sequences for PKA (3-5). The basic consensus phosphorylation sites for this enzyme are Arg-Arg-X-(Ser/Thr), (Arg/Lys)-X-X-(Ser/Thr), and (Arg/Lys)-X-(Ser/ Thr) (3, 4, 6, 7).The molecular recognition of the peptide sequence surrounding the phosphorylated residue of a substrate usually is referred to as the "peptide specificity" of a protein kinase. The phosphorylated residue is conventionally named P0, the substrate residues immediately N-and C-terminal to the P0 position as PϪ1 and Pϩ1, r...

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Section: Resultsmentioning

confidence: 89%

“…In our laboratory, we have demonstrated the participation of the peptide substrate in the activation of a fungal (Mucor rouxii) holoenzyme by cAMP (29). It was interesting to analyze whether the substrate could participate in the PKA holoenzyme activation in S. cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

Galello

Portela

Moreno

et al. 2010

Journal of Biological Chemistry

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“…However, even though the WT extracts showed cAMPdependent phosphorylation of Kemptide, the phosphorylation of the Pyk recombinant proteins was cAMP-independent. This lack of dependence on cAMP could be explained if PKA from S. cerevisiae behaves as PKA from the fungus Mucor rouxii, where we have demonstrated (12) that the protein substrate is involved in the activation of PKA by cAMP and that, depending on the incubation conditions, different degrees of activation by cAMP are obtained using different substrates. In fact, under the phosphorylation conditions used for Fig.…”

Section: Discussionmentioning

confidence: 97%

“…The main objective of our group is to study the mechanism of in vivo activation of PKA by cAMP and the participation of the substrate in this process using lower eukaryotic models (11)(12)(13)(14). While looking for PKA substrates in S. cerevisiae, we found that both isoforms of pyruvate kinase, Pyk1 and Pyk2, are substrates of PKA.…”

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confidence: 97%

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Portela

Howell²,

Moreno

et al. 2002

Journal of Biological Chemistry

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Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1⅐Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1 w1 ). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n H value of 1.4, as compared with an n H of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

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“…rouxii PKA holoenzyme was prepared from mycelial powder through DEAE-cellulose and (NH 4 ) 2 SO 4 precipitation and the C subunit was isolated by dissociating sucrose gradient centrifugation with 0.5 M NaCl and 1 mM cAMP as described previously (Zaremberg et al 2000). Phosphotransferase activity of the M.rouxii C subunit was assayed in a reaction mixture containing 50 mM Tris-HCl pH 7.4, 0.1 mM [c 32 P]ATP (300 cpm/pmol), 0.1 mM EGTA, 0.1 mM EDTA, 15 mM MgCl 2 , 10 mM b-mercaptoethanol and variable amounts of the two substrate peptides assayed: kemptide (LRRASLG) and rhotide (IR-RNSQKFV), which were synthesized by BioSynthesis Inc.. After 15-min incubation at 30°C aliquots from each reaction were processed by the phosphocellulose paper method (Roskoski 1983).…”

Section: Kinetic Analysismentioning

confidence: 99%

Mucor rouxii Rho1 protein; characterization and possible role in polarized growth

Argimón

Galello²,

Pereyra³

et al. 2006

Antonie van Leeuwenhoek

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We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70-85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the beta-1,3-glucan synthase complex in fungi in which beta-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of beta-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of beta-1,3-glucans on the regulation of morphology of zygomycetes.

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Mechanism of Activation of cAMP-Dependent Protein Kinase: In Mucor rouxii the Apparent Specific Activity of the cAMP-Activated Holoenzyme Is Different than That of Its Free Catalytic Subunit

Cited by 12 publications

References 31 publications

Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Mucor rouxii Rho1 protein; characterization and possible role in polarized growth

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