Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain.
Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.
Lengyel
I
I
Institute of Biochemistry, Biological Research Center, Szeged, Hungary. ilengyel@nucleus.szbk.u-szeged.hu
Nairn
A
A
McCluskey
A
A
Tóth
G
G
Penke
B
B
Rostas
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010455:Peptides; C085903:autocamtide-2; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517599:Camk1 protein, rat; C517686:Camk4 protein, rat; C517682:Pnck protein, rat