Phosphorylation of rabbit skeletal muscle phosphorylase kinase by cyclic GMP‐dependent protein kinase

Cohen, Philip

doi:10.1016/0014-5793(80)80277-8

Cited by 37 publications

(5 citation statements)

References 34 publications

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“…With δ being present in both rPhK and α γδ , their activities were, as expected, Ca 2+ -dependent; both complexes required the same concentration of Ca 2+ as RM PhK to achieve full activation, namely, 5.5 μ M, in assays carried out at pH 8.2 (Figure 5A). The activating concentration of Ca 2+ observed for the recombinant complexes was also in close agreement with that reported for full activation of RM PhK at pH 8.2 in previous studies using alternative methods (34, 41–43). The shapes of the Ca 2+ -titration curves for the recombinant enzymes were also very similar to that for RM PhK (Figure 5A).…”

Section: Resultssupporting

confidence: 90%

Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

et al. 2009

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Understanding the regulatory interactions among the 16 subunits of the (αβγδ)4 phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells α, β, γ, and δ subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding αγδ trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and αγδ exhibited only a very low Ca2+-independent activity and a Ca2+-dependent activity similar to that of the native holoenzyme with [Ca2+]0.5 of 0.4 μM for the RM PhK, 0.7 μM for the rPhK, and 1.5 μM for the αγδ trimer. The RM PhK, rPhK, and αγδ subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the α–γ intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant αγδ subcomplex. Our results indicate that both the rPhK and αγδ subcomplex are promising models for future structure–function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.

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Section: Resultssupporting

confidence: 90%

Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

et al. 2009

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“…In addition protein phosphatase 1 accounts for > 90 of the activity towards the /?-subunit of phosphorylase kinase [34-361, the site that determines the activity of the enzyme [37]. Since insulin does not alter the phosphorylation state of phosphorylase, and only has a slight effect on phosphorylase kinase (Table I), it could be argued that activation of protein phosphatase 1 or protein phosphatase 2A cannot underlie the dephosphorylation of glycogen synthase by insulin, otherwise corresponding changes in the activity of phosphorylase or phosphorylase kinase would be observed.…”

Section: Effgect Of Diabetes On the Kinetic Properties And Pliosphalementioning

confidence: 99%

Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo

Parker

Caudwell

Cohen

1983

European Journal of Biochemistry

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278

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Rabbits were starved for 24 h and injected with propranolol ( 2 mg/kg) with or without insulin (33 pg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol-treated animals the activity ratio (F glucose 6-phosphate) of glycogen synthase was 0.18 0.02 and the K, for glucose 6-phosphate was 1.2 0.1 mM. In (propranolol + insuIin)-treated animals the activity ratio was 0.35 & 0.02 and the I(, for glucose 6-phosphate was 0.60 & 0.05 mM.0.09 niol phosphate/mol subunit, whereas this value was 2.33 0.09 mol phosphate/mol subunit from (propranolol + insulin)-treated animals.The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)-treated animals there was a decrease of 0.4-0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (la, lb, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin.Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in v i m at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed.The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic-AMPdependent protein kinase (la, I b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase.Neither 24-h starvation, nor alloxon-induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.Glycogen synthase purified to homogeneity from propranolol-treated animals contained 2.74Glycogen synthase is the best documented and one of the most complex examples of 'multisite phosphorylation', a phenomenon that is being encountered with increasing frequency [I]. The enzyme is phosphorylated on seven serine residues by at least five different protein kinases [2] [7].In general, phosphorylation increases the K , for UDPglucose, decreases the Ki for inhibitors such as Pi and ADP, and increases the K, for the activator glucose-6-P [S]. Phosphorylation of sites (3a + 3b + 3c) or site 2 influences the kinetic parameters to a greater extent than site l a , but the ~ Abbreviations. SDS, sodium dodecyl sulphate; HEDTA, N '-(2-hydroxyethyl)-ethylenedianiine-N,N',N-triacetate; HPLC, high-performance liquid chromatography ; glucose-6-P, glucose 6-phosphate.Enzymes. Phosphorylase (EC 2.4.1.1); phosphorylase kina...

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“…This result holds from concentrations of each ranging from 50 nM to 2 pMand at pH 6.0 and pH 7.2 (not shown). Thus, tyrosine hydroxylase represents a relatively good substrate for cyclic GMP-dependent protein kinase since many other proteins are phosphorylated by a factor of 20-25-fold less efficiently (Lincoln and Corbin, 1977, 19784. Other protein substrates that are phosphorylated in vitro with similar kinetics by cyclic GMP-and cyclic AMP-dependent protein kinases include the cerebellar protein G-substrate (Aswad and Greengard, 1981), DARPP-32 (Hemmings et al, 1984), high-mobility group protein 14 (Walton et al, 1982), histone H2B (Glass and Krebs, 1979), histone H1 (Zeilig et al,198 l), troponin-I (Lincoln and Corbin, 1978b;Blumenthal et al, 1978), protein S6 and other eukaryotic ribosomal proteins (Issinger et al, 1980;Del Grande and Traugh, 1982), various forms of myosin light chain kinases (Nishikawa et al, 1984), and phosphorylase kinase (Cohen, 1980). In some of these cases, each protein kinase phosphorylated the same amino acid residue, but in several instances unique sites were phosphorylated by one or the other enzyme or each kinase phosphorylated multiple sites with differential initial rates.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP‐Dependent Protein Kinase

Roskoski

Vulliet

Glass

1987

Journal of Neurochemistry

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Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.

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Phosphorylation of rabbit skeletal muscle phosphorylase kinase by cyclic GMP‐dependent protein kinase

Cited by 37 publications

References 34 publications

Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP‐Dependent Protein Kinase

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