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Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain
I Lengyel1, A Nairn, A McCluskey
1Institute of Biochemistry, Biological Research Center, Szeged, Hungary. ilengyel@nucleus.szbk.u-szeged.hu
Abstract:
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.
Insights
A peptide fragment (F12) from the ATP-binding domain inhibits Ca(2+)/calmodulin-dependent protein kinase II (CaMPK-II) activity. This suggests intramolecular interactions within the ATP-binding domain regulate CaMPK-II.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ca(2+)/calmodulin-dependent protein kinase II (CaMPK-II) is crucial for physiological processes.
- CaMPK-II requires Ca(2+)/CaM binding for activation.
- The inactive form of CaMPK-II does not bind ATP, implying intramolecular interactions involving the ATP-binding domain.
Purpose of the Study:
- To investigate the role of the ATP-binding domain in CaMPK-II regulation.
- To characterize the inhibitory effect of a peptide fragment from the CaMPK-II ATP-binding domain.
Main Methods:
- Enzyme inhibition assays using a peptide fragment (F12) of the CaMPK-II ATP-binding domain.
- Kinetic analysis of inhibition with respect to substrates (autocamtide-2) and ATP.
- Testing F12 inhibition against related kinases (CaMPK-I, CaMPK-IV, PKA).
Main Results:
- The F12 peptide (CaMPK-II(23-34)) inhibited Ca(2+)/CaM-dependent CaMPK-II activity (IC50 = 3 microM) but not Ca(2+)/CaM-independent activity.
- F12 exhibited mixed inhibition kinetics with respect to autocamtide-2 and ATP.
- F12 showed specificity for CaMPK-II, with weaker inhibition of CaMPK-I and minimal inhibition of CaMPK-IV and PKA.
Conclusions:
- The ATP-binding domain of CaMPK-II is involved in intramolecular interactions regulating its activity.
- The F12 peptide fragment provides insights into the molecular mechanisms of CaMPK-II regulation.
- These findings highlight the potential for targeting the ATP-binding domain for kinase modulation.