The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands.
The C terminus (ct) of protein kinase C-alpha (PKCalpha) has a type I PDZ binding motif, whereas GluR2 has a type II PDZ binding motif. Both motifs are recognized by the PDZ domain of protein interacting with protein kinase C (PICK1), and PICK1-PKCalpha-controlled phosphorylation regulates the synaptic expression and function of GluR2. Here, we show that a specific mutation within the carboxylate-binding loop of the PDZ domain of PICK1 (K27E; PICK1-KE) results in a loss of interaction with GluR2 but not with PKCalpha. In GST pull-down studies, PICK1-WT (wild type) but not PICK1-KE was retained by GST-ct-GluR2. Furthermore, PICK1-WT co-immunoprecipitated both PKCalpha and GluR2, whereas PICK1-KE only co-immunoprecipitated PKCalpha. In heterologous cells, PICK1-WT, but not PICK1-KE, clustered GluR2 and also clustered GluR1 in a GluR2-dependent manner. However, neither PICK1-WT nor PICK1-KE altered the distribution of PKCalpha, even after phorbol ester-induced redistribution of PKCalpha to the membrane. Finally, PICK1-KE showed no mislocalization when compared with PICK1-WT in neurons. Taken together, it appears that the PDZ domain of PICK1 is less sensitive to mutations for PKCalpha when compared with GluR2 binding. These results suggest that the PDZ domain of PICK1 has distinct PKCalpha and GluR2 binding subsite(s).
Dev
Kumlesh K
KK
Medical Research Council Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol BS8 1TD, UK. kumlesh_k.dev@pharma.novartis.com
Nakanishi
Shigetada
S
Henley
Jeremy M
JM
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008024:Ligands; D009154:Mutation; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D018091:Receptors, AMPA / Q000378:metabolism*; D014162:Transfection
D002352:Carrier Proteins; D008024:Ligands; D009687:Nuclear Proteins; C498297:PICk1 protein, human; D018091:Receptors, AMPA; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C104722:glutamate receptor ionotropic, AMPA 2