The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase II Delta (CaMKIIδ) Phosphorylates Titin N2B and PEVK Spring Elements

Abstract

Titin is phosphorylated by several kinases that significantly affect titin mechanical properties. PKA and PKG phosphorylates titin N2B element and decreases titin's stiffness, while PKCα phosphorylates titin PEVK spring element and increases titin's stiffness. CaMKII is a Ca2+ and calmodulin dependent serine/threonine kinase that is activated by increases in cellular Ca2+. Four isoforms have been described (α, β, γ, and δ). CaMKII delta (CaMKIIδ) is the predominant isoform in the heart. It has been found that increases in CaMKIIδ activity causes severe left ventricular dysfunction. The aim of the present study is to determine whether CaMKIIδ phosphorylates titin and, if so, identify its target domains. To determine whether CaMKIIδ phosphorylates titin in mouse left ventricular muscle, LV skinned fibers were incubated with recombinant human CaMKIIδ expressed in insect cells and [γ-32P]ATP. We found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers. Further, pre-incubation of the LV skinned fiber with protein phosphatase 1 (PP1) significantly increases 32P incorporation on titin. To identify the CaMKIIδ titin target domains, human titin N2B, PEVK, Ig8-15, I27-34, and murine N2B recombinant proteins expressed in E. coli were incubated with CaMKIIδ and [γ-32P]ATP. We found that titin N2B and PEVK spring elements, but not Ig domains, were phosphorylated by CaMKIIδ. Prediction of the specific phosphorylation sites suggest overlap with the PKC and PKA sites (including S170 of the PEVK element a PKC substrate) and work to indentify the specific substrate sites and the possible effect on mechanics is in progress. In summary, the present study showed that CaMKIIδ phosphorylates titin spring elements.