CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

Jaqueline R. Kulbe,Isabelle Buard,Mark L. Dell'Acqua,K. Ulrich Bayer,Steven J. Coultrap

doi:10.1074/jbc.m109.069351

Jaqueline R. Kulbe, Isabelle Buard + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.069351

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Abstract

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

Highlights

Phosphorylation in the activation loop is a necessary step to generate full activity of many kinases, including PKA, PKC, and several CaMKs
Ca2ϩ/CaM Stimulates Phosphorylation of Endogenous Tyrosine Hydroxylase by Autonomous CaMKII within PC12 Cells— The biochemical data clearly demonstrated that autonomous CaMKII activity can be further stimulated by Ca2ϩ/CaM. Is this biologically relevant, or are cellular substrates already maximally phosphorylated by the unstimulated autonomous activity, especially when autonomy acts over prolonged periods of time? To address this question, we examined phosphorylation of tyrosine hydroxylase (TH) at Ser-19 in differentiated PC12 cells expressing various GFP-CaMKII mutants, before and after
Generation of CaMKII autonomy by Thr-286 autophosphorylation is a vital step in regulating synaptic plasticity and learning/memory [15]

Summary

Regulation of Autonomous CaMKII Activity

Proteins were phosphorylated in the same buffer as peptides, but substrate concentration was. Against CaMKII␣ (CB␣2), all CaMKII isoforms (BD Pharmin- CaM still stimulated Thr-286-prephosphorylated CaMKII gen) phospho-T286 or -T305 CaMKII, GluR1 phospho-S831 more than 4 fold over autonomous activity, to the same level as (PhosphoSolutions), total GluR1 (Calbiochem), or NR2B phos- stimulated activity of naïve kinase, at least for syntide (Fig. 1C). In these assays, CaMKII concentration was increased, as the four peptides were poor substrates compared with AC2 and syntide (see supplemental Fig. S2). For all regular substrates tested, autonomous CaMKII activity can be further stimulated 4 – 6 ence AC2 is derived the mutations did not decrease autonomy toward syntide (Fig. from the CaMKII autoregulatory region around Thr-286, and 3D) or the other regular substrate peptides (supplemental can bind at the T-site (T-substrate)(29)(see Fig. Fig. High CaMKII Autonomy Toward the Cellular T-substrate NR2B—Other than the Thr-286 autophosphorylation

Aut ϭ

Findings

DISCUSSION