Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation

Abstract

The activity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) depends on the balance between activating and inhibitory autophosphorylation (Thr 286 and Thr 305/306, respectively, in the human a isoform). Variation in the lengths of the flexible linkers that connect the kinase domains of CaMKII to a central oligomeric hub could alter transphosphorylation rates within a holoenzyme, thereby affecting the balance of autophosphorylation outcomes. Using a singlemolecule assay for visualization of CaMKII phosphorylation on glass, we show that the balance of autophosphorylation is flipped between CaMKII-a and CaMKII-b, the two principal isoforms in the brain. CaMKII-a, with a ~30 residue kinase-hub linker, readily acquires activating autophosphorylation, which we show is resistant to removal by phosphatases. CaMKII-b, with a ~200 residue kinase-hub linker, is biased towards inhibitory autophosphorylation. Thus, the responsiveness of CaMKII to calcium signals can be tuned by varying the relative levels of the a and b isoforms.

Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase

“…We activated CaMKII-α in the diluted cell lysate with saturating amounts of Ca 2+ /CaM (5 µM) and ATP (10 mM), captured the activated CaMKII-α on a coverslip functionalized with streptavidin, and measured the distribution of fluorescence intensity of the imaged spots. These conditions have been shown previously to result in robust phosphorylation of CaMKII-α on Thr 286, with a lower amount of phosphorylation on Thr 305/Thr 306 (Bhattacharyya et al 2020).…”

“…The calmodulin-binding element contains two residues, Thr 305 and Thr 306, that can be phosphorylated when CaMKII is activated, and phosphorylation at these sites prevents rebinding of Ca 2+ /CaM to the holoenzyme ( Colbran, 1993 ; Bhattacharyya et al, 2020b ). We tested the stability of the hub in the presence of variants of Peptides A and B ( Figure 1C ) in which Thr 305 is phosphorylated (hereafter referred to as Peptide A phos and Peptide B phos , respectively).…”

“…We studied the effect of activation on the stability of the intact CaMKII-α holoenzyme expressed in mammalian cells, using TIRF microscopy and single-molecule analysis. We developed a cell-based assay that allowed us to rapidly isolate CaMKII-α after cell lysis, while reducing the heterogeneity that can result from proteolysis or the aggregation that occurs during purification ( Bhattacharyya et al, 2020b ). Briefly, CaMKII-α was tagged at the N-terminus with a biotinylation tag and mEGFP ( Cormack et al, 1996 ; Zacharias et al, 2002 ) and overexpressed in HEK293T cells.…”

“…Subunit exchange in CaMKII has been proposed originally as a mechanism whereby the activation states of CaMKII could be maintained indefinitely, contributing to the storage of memories ( Lisman, 1994 ). It is difficult to reconcile such a mechanism with the action of phosphatases, which are expected to reset the activation sates of CaMKII ( Lee et al, 2009 ; Bhattacharyya et al, 2020b ). Instead, we speculate that the destabilization of CaMKII holoenzymes upon activation is a mechanism that potentiates the effects of Ca 2+ pulses under conditions when calmodulin is limiting, as is the case in neurons ( Persechini and Stemmer, 2002 ; Pepke et al, 2010 ).…”

“…The calmodulin-binding element contains two residues, Thr 305 and Thr 306, that can be phosphorylated when CaMKII is activated, and phosphorylation at these sites prevents rebinding of Ca 2+ /CaM to the holoenzyme (Colbran 1993;Bhattacharyya et al 2020).We tested the stability of the hub in the presence of variants of Peptides A and B ( Figure 1C) in which Thr 305 is phosphorylated (hereafter referred to as Peptide A phos and Peptide B phos , respectively). In these experiments, CaMKII-α hub at a final subunit concentration of 120 µM was incubated with Peptide A phos for five minutes at room temperature prior to injection into the mass spectrometer under similar ionization conditions as before.…”