Ca2+/Calmodulin-dependent Protein Kinase II Binds to and Phosphorylates a Specific SAP97 Splice Variant to Disrupt Association with AKAP79/150 and Modulate α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor (AMPAR) Activity

Steven J. Tavalin,Roger J. Colbran,Yuxia Jiao,Anthony J. Baucum,Yelyzaveta A. Nikandrova

doi:10.1074/jbc.m109.033985

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes trafficking and activation of the GluR1 subunit of alpha-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) during synaptic plasticity. GluR1 is also modulated in parallel by multiprotein complexes coordinated by synapse-associated protein 97 (SAP97) that contain A-kinase anchoring protein 79/150 (AKAP79/150), protein kinase A, and protein phosphatase 2B. Here we show that SAP97 is present in CaMKII immune complexes isolated from rodent brain as well as from HEK293 cells co-expressing CaMKIIalpha and SAP97. CaMKIIalpha phosphorylated recombinant SAP97 within immune complexes in vitro and in intact cells. Four alternative mRNA splice variants of SAP97 expressing combinations of four inserts (I2, I3, I4, I5) in the U5 region between Src homology 3 (SH3) and guanylyl kinase-like (GK) domains were identified in rat brain at postnatal day 21. CaMKIIalpha preferentially phosphorylated a full-length SAP97 and a glutathione S-transferase (GST) fusion protein containing the I3 and I5 inserts (SAP97-I3I5 and GST-SH3-I3I5-GK, respectively) and also specifically interacted with GST-SH3-I3I5-GK compared with GST proteins containing other naturally occurring insert combinations. AKAP79/150 also directly and specifically bound only to GST-SH3-I3I5-GK, but CaMKII phosphorylation of GST-SH3-I3I5-GK prevented this interaction. AKAP79-dependent down-regulation of GluR1 AMPAR currents was ablated by overexpression of SAP97-I2I5 (which does not bind AKAP79) or by infusion of active CaMKIIalpha. Collectively, the data suggest that CaMKIIalpha targets a specific SAP97 splice variant to disengage AKAP79/150 from regulating GluR1 AMPARs, providing new insight into protein-protein interactions and phosphorylation events that are required for normal regulation of glutamatergic synaptic transmission, learning, and memory.

Highlights

Synaptic plasticity, learning, and memory require a precisely orchestrated series of molecular changes in dendritic spines, including changes in protein-protein interactions and posttranslational modifications
Normal synaptic plasticity requires changes in phosphorylation of Ser-831 and Ser-845 in the GluR1 subunit of the AMPAtype glutamate receptor (AMPAR) (8 –16). Phosphorylation of these sites by calmodulin-dependent protein kinase II (CaMKII) or protein kinase C (PKC) and by protein kinase A (PKA), respectively, directly enhances AMPAR activity and is required for long term potentiation, whereas long term depression requires dephosphorylation of these sites mediated by protein phosphatase 2B (PP2B) and/or protein phosphatase 1
An excess of the classical substrate pep- synapse-associated protein 97 (SAP97) Splicing and CaMKII Modulate AKAP79-dependent tide, syntide-2, had no effect on binding of Thr-286-autophos- Regulation of GluR1 AMPARs—Interaction of AKAP79/150 phorylated CaMKII to glutathione S-transferase (GST)-Src homology 3 (SH3)-I3I5-guanylyl kinase-like (GK) in slot-blot assays with SAP97 targets PKA and PP2B to GluR1 to bidirectionally

Summary

Introduction

Synaptic plasticity, learning, and memory require a precisely orchestrated series of molecular changes in dendritic spines, including changes in protein-protein interactions and posttranslational modifications. CaMKII phosphorylation of Ser-39 in the N-terminal L27 domain of SAP97 modulates trafficking of SAP97 and the associated proteins (Fig. 1) (22, 24, 25).

Results

Conclusion