Studies on the specificity of phosphorylase kinase using peptide substrates (original) (raw)
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Comparison of Substrate Specificities of Protein Kinases A and C Based on Peptide Substrates
Bioorganic Chemistry, 1994
Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAEcellulose, precipitation with (NH14)2S04 and gel filtration on Sephadex G}200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase bt, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the a-subunit or the fl-subunit), histone HI and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, H and III. Protein phosphatase'I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3 % of the phosphorylase kinase phosphatase and phosphorylaae phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-l, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95°% of the activity towards the a-subunit of phosphorylase kinase that was recovered from Sephadex G0200, It accounted for 10-15% of the glycogen synthase phosphataseo1 and glycogen synthase phosphatase2 activity, but less than 5 % of the activity against the 1subunit of phosphorylase kinase and 1-2 % of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95 % of the phosphorylase phosphatase and 1)-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5 % of the a-phosphorylase kinase phosphatase activity. Protein phosphatast-IH was sometimes eluted from Sephadex G-200 as a species of apparent mol.wt. 75000 (termed IIIA), sometimes as a species of mol.wtb 46000 (termed IuB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-4lA and IIIB were identical. These findings, taken with the observation that phosphorylase phosphatase, 1-phosphorylase kinase phosphatase, glycogen synthase phosphatasedI and glycogen synthase phos. phatase-2 activities co-purified up to the Sephadex G0200 step, suggest that a single protein phosphatase (protein phosphatase-HI) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G. A. & Antoniw, J. F. (1977) Btochem. J. 162, 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III. Ever since the discovery that cyclic AMP-skeletal-miuscle phosphorylase kinase (EC 2.7.1.37) dependent protein kinase (EC2.7.1.37) activates and inactivates skeletal-muscle glycogen synthase (EC 2.4.1.11) (Soderling et al., 1970), there has been
Journal of Biological Chemistry, 1996
The C terminus of the catalytic ␥-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the ␥⅐␦ complex. Antibodies raised against the very C terminus of the ␥-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the ␥⅐␦ complex, in the presence and absence of Ca 2؉ . In the presence of Ca 2؉ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the ␥⅐␦ complex 2.5-fold. Activation of the holoenzyme and the ␥⅐␦ complex by anti-PhK5 was 50 -70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the ␥⅐␦ complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the ␥-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.
Archives of Biochemistry and Biophysics, 1999
Residues 302-326 of the catalytic (␥) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhK␥ was expressed using Escherichia coli. This yielded two stable, high-activity PhK␥ forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhK␥ (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhK␥ residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact ␥ subunit. This implied that the lack of 25 PhK␥ Cterminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhK␥ that consisted of residues 1-300, denoted ␥1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.
European journal of biochemistry / FEBS, 1990
The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-t...
Journal of Neurochemistry, 1991
The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the K, and V,,, of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 pLM and 7.1 pmol/ min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high K, of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydrox ylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to seine 40. Tyrosine hydroxylase (36-46), for example, exhibited a K, of 108 pM and a V,,, of 6.93 pmol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine I9 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme. Key Words: Tyrosine hydroxylase-Cyclic AMP-dependent protein kinase-Protein serine kinases-Substrate specificity-Pyruvate kinase-6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Roskoski R. Jr. and Ritchie P. Phosphorylation of rat tyrosine hydroxylase and its model peptides in vitro by cyclic AMPdependent protein kinase.
Substrate specificity of phosphorylase kinase: Effects of heparin and calcium
Archives of Biochemistry and Biophysics, 1987
Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2'; the rate of syn tide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the V