scholarly journals The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase

1977 ◽  
Vol 161 (2) ◽  
pp. 371-382 ◽  
Author(s):  
A J G Moir ◽  
H A Cole ◽  
S V Perry
Keyword(s):  
Troponin T ◽  
Troponin C ◽  
Amino Acid Sequences ◽  
The Other ◽  
Phosphorylase Kinase ◽  
Phosphorylation Sites ◽  
Native Protein ◽  
Dependent Protein Kinase ◽  
Troponin Complex ◽  
Dependent Protein

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3′:5′-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx, SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser, SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.

scholarly journals Phosphorylation of troponin and the effects of interactions between the components of the complex

1974 ◽  
Vol 141 (3) ◽  
pp. 733-743 ◽  
Author(s):  
Samuel V. Perry ◽  
Heather A. Cole
Keyword(s):  
Protein Kinase ◽  
Troponin I ◽  
Troponin T ◽  
Troponin C ◽  
Phosphorylase Kinase ◽  
Significant Extent ◽  
Dependent Protein Kinase ◽  
Prolonged Incubation ◽  
Troponin Complex ◽  
Dependent Protein

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3′:5′-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca2+sensitivity on the adenosine triphosphatase of densensitized actomyosin.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

scholarly journals Phosphorylation of skeletal-muscle troponin I and troponin T by phospholipid-sensitive Ca2+-dependent protein kinase and its inhibition by troponin C and tropomyosin

1984 ◽  
Vol 218 (2) ◽  
pp. 361-369 ◽  
Author(s):  
G J Mazzei ◽  
J F Kuo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin T ◽  
Troponin C ◽  
Cardiac Troponin I ◽  
Equimolar Amount ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Skeletal-muscle troponin I and troponin T were found to be rapidly phosphorylated by cardiac phospholipid-sensitive Ca2+-dependent protein kinase, with Km values of 6.66 and 0.13 microM respectively. Stoichiometric phosphorylation of skeletal troponin I (endogenous phosphate content 0.7 mol/mol) indicated that the Ca2+-dependent enzyme and cyclic AMP-dependent protein kinase incorporated 0.9 and 0.8 mol/mol respectively. The same experiments with skeletal troponin T (endogenous phosphate content 1.9 mol/mol) revealed a maximal phosphorylation of 2 mol/mol by the Ca2+-dependent enzyme, whereas the cyclic AMP-dependent enzyme was unable to phosphorylate troponin T. The Ca2+-dependent enzyme phosphorylated both serine and threonine residues in skeletal and cardiac troponin I or troponin T; the cyclic AMP-dependent enzyme, in comparison, phosphorylated only serine in skeletal and cardiac troponin I. Although an equimolar amount of skeletal or cardiac troponin C markedly inhibited (80-90%) phosphorylation of skeletal and cardiac troponin I by the Ca2+-dependent enzyme, these troponin C preparations inhibited only phosphorylation of skeletal troponin I, but not that of cardiac troponin I, by the cyclic AMP-dependent enzyme. Calmodulin and Ca2+-binding protein S-100a could mimic the inhibitory effect of troponin C. A tissue specificity appeared to exist for the skeletal troponin T-skeletal troponin C interaction. Inhibition of troponin T phosphorylation by an equimolar amount of troponin C was lower than that of troponin I phosphorylation; these findings might explain in part why troponin T was the major substrate for the Ca2+-dependent enzyme in the troponin complex. The present studies indicate that skeletal and cardiac troponin I and troponin T were effective substrates for phospholipid-sensitive Ca2+-dependent protein kinase, suggesting a potential involvement of this Ca2+-effector enzyme in the regulation of myofibrillar activity.

scholarly journals Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

1999 ◽  
Vol 342 (2) ◽  
pp. 287-292 ◽  
Author(s):  
Antonio CASAMAYOR ◽  
Nick A. MORRICE ◽  
Dario R. ALESSI
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
The Other ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Equivalent Position ◽  
293 Cells ◽  
Dependent Protein ◽  
Kinase Substrates

3-Phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimulated 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and Ser-410 and the level of phosphorylation of each site was unaffected by stimulation with insulin-like growth factor-1. Mutation of Ser-241 to Ala abolished PDK1 activity, whereas mutation of the other phosphorylation sites individually to Ala did not affect PDK1 activity. Ser-241, unlike the other phosphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A1. Ser-241 lies in the activation loop of the PDK1 kinase domain between subdomains VII and VIII in the equivalent position to the site that PDK1 phosphorylates on its protein kinase substrates. PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.

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