Phosphorylation dependent regulation of CaMKII gamma activity and its implication in vascular smooth muscle function

Abstract

Calcium/calmodulin dependent kinase II (CaMKII) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue (Thr²⁸⁷) within each subunit of its oligomeric structure. Using an antisense oligonucleotide, CaMKII γ was previously demonstrated to lead to decreased contractility of blood vessels. However, the mechanism of this process remains unknown.

In this thesis. I show that the six CaMKII γ smooth muscle variants are differentially targeted when expressed in cultured cells. I also show that CaMKII γ variants disrupt microtubules upon Ca²⁺ stimulation in a variant-independent manner. Similar microtubule disassembly was shown in A7r5 vascular smooth muscle cells which display CaMKII properties similar to blood vessels. The major discovery of this thesis is the determination of a novel and physiologically significant phosphorylation-dependent function of CaMKII γ. Phosphorylation of Ser²⁶ was found to switch off kinase activity. I show that phosphorylation of CaMKII gamma at Ser ²⁶, a residue located within the ATP binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, a phosphospecific Ser²⁶ antibody was generated and I demonstrated an increase of Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, the Ser²⁶ residue was mutated into an Ala or Asp. The recombinant S26D mutant mimicking the phosphorylated state of CaMKII displays a dramatic decrease in Thr²⁸⁷ autophosphorylation levels in vitro and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), while the S26A mutation has no effect. These data combined with molecular modeling indicate that a negative charge at Ser²⁶ of CaMKII gamma inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most likely by blocking ATP binding. Here I propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity, which may play an important role in vascular contractility.