Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5

Oscar Cerda,James S. Trimmer

doi:10.1074/jbc.m111.251942

Oscar Cerda, James S. Trimmer

Open Access

https://doi.org/10.1074/jbc.m111.251942

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Abstract

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

Highlights

cyclin-dependent kinase 5 (CDK5) Directly Phosphorylates Kv2.1—To address whether Kv2.1 is a direct substrate of CDK5-p35, we performed in vitro phosphorylation reactions using purified recombinant human CDK5-p35 complex and a bacterially expressed recombinant GST fusion protein containing a fragment of the Kv2.1 cytoplasmic C terminus corresponding to amino acids 509 – 853, which contains a total of 49 Ser and 24 Thr residues, and 11 of the sites (Fig. 1B) identified in our previous analysis of in vivo Kv2.1 phosphorylation sites [15]
It is possible that cyclin-dependent kinase 5 (CDK5) overexpression plays an indirect role in increasing Kv2.1 phosphorylation by regulating other protein kinases (PKs) and/or PPs that directly determine the Kv2.1 phosphorylation state
To determine whether any of the effects of manipulating CDK5 activity on Kv2.1 phosphorylation are mediated through PP1, we investigated the level of Kv2.1 phosphorylation in HEK293 cells co-expressing Kv2.1 and CDK5-p35, together with either WT PP1 or the PP1(T320A) mutant that is refractory to CDK5-dependent inhibition

Summary

Introduction

CDK5 Directly Phosphorylates Kv2.1—To address whether Kv2.1 is a direct substrate of CDK5-p35, we performed in vitro phosphorylation reactions using purified recombinant human CDK5-p35 complex and a bacterially expressed recombinant GST fusion protein containing a fragment of the Kv2.1 cytoplasmic C terminus corresponding to amino acids 509 – 853, which contains a total of 49 Ser and 24 Thr residues, and 11 of the sites (Fig. 1B) identified in our previous analysis of in vivo Kv2.1 phosphorylation sites [15]. We found that roscovitine treatment of cultured hippocampal neurons leads to a loss of pS603 signal on immunoblots as well as a slightly increased electrophoretic mobility of the total Kv2.1 pool, consistent with a reduction in overall phosphorylation state (Fig. 3E).

Results

Conclusion