Abstract
The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.
Highlights
A characteristic feature of signaling by protein kinases is that the kinases are themselves regulated by phosphorylation
The activity of calmodulindependent protein kinase II (CaMKII) is modulated by autophosphorylation on two sites, Thr 286 and Thr 305/306, both located in a regulatory segment that immediately follows the kinase domain (Figure 1a,d)
CaMKII is unusual among protein kinases because it is assembled into a large holoenzyme with twelve to fourteen subunits
Summary
A characteristic feature of signaling by protein kinases is that the kinases are themselves regulated by phosphorylation. For most kinases, this involves the phosphorylation of one or more residues in the activation loop, a regulatory element located at the active site of the kinase (Huse and Kuriyan, 2002; Johnson et al, 1996; Nolen et al, 2004). The activity of CaMKII is modulated by autophosphorylation on two sites, Thr 286 and Thr 305/306 (residue numbering corresponds to the sequence of human CaMKII-a), both located in a regulatory segment that immediately follows the kinase domain (Figure 1a,d).
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