Two Adaptor Proteins Differentially Modulate the Phosphorylation and Biophysics of Kv1.3 Ion Channel by Src Kinase

• Published in: The Journal of biological chemistry Vol. 277; no. 15; pp. 13268 - 13280
• Main Authors: Cook, Karen K., Fadool, Debra A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.04.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line; GRB10 Adaptor Protein; Grb10 protein; Humans; Kv1.3 Potassium Channel; Mutagenesis, Site-Directed; n-Shc protein; Neuropeptides - physiology; Olfactory Bulb - metabolism; Phosphorylation; Potassium Channels - genetics; Potassium Channels - metabolism; Potassium Channels, Voltage-Gated; Proteins - physiology; Rats; Rats, Sprague-Dawley; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 3; src-Family Kinases - metabolism; v-src gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108898200

The Shaker family K+channel protein, Kv1.3, is tyrosine phosphorylated by v-Src kinase at Tyr137 and Tyr449 to modulate current magnitude and kinetic properties. Despite two proline rich sequences and these phosphotyrosines contained in the carboxyl and amino terminals of the channel, v-Src kinase fails to co-immunoprecipitate with Kv1.3 as expressed in HEK 293 cells, indicating a lack of direct Src homology 3- or Src homology 2-mediated protein-protein interaction between the channel and the kinase. We show that the adaptor proteins, n-Shc and Grb10, are expressed in the olfactory bulb, a region of the brain where Kv1.3 is highly expressed. In HEK 293 cells, co-expression of Kv1.3 plus v-Src with Grb10 causes a decrease in v-Src-induced Kv1.3 tyrosine phosphorylation and a reversal of v-Src-induced Kv1.3 current suppression, increase in inactivation time constant (τinact), and disruption of cumulative inactivation properties. Co-expression of Kv1.3 plus v-Src with n-Shc did not significantly alter v-Src-induced Kv1.3 current suppression but reversed v-Src induced increased τinact and restored the right-shifted voltage at half-activation (V1/2) induced by v-Src. The v-Src-induced shift in V1/2 and increased τinact was retained when Tyr220, Tyr221, and Tyr304 in the CH domain of n-Shc were mutated to Phe (triple Shc mutant) but was reversed back to control values when either wild-type Shc or the family member Sck, which is not a substrate for Src kinase, was substituted for the triple Shc mutant. Thus the portion of the CH domain that includes Tyr220, Tyr221, and Tyr304 may regulate a shift in Kv1.3 voltage dependence and inactivation kinetics produced by n-Shc in the presence of v-Src. Collectively these data indicate that Grb10 and n-Shc adaptor molecules differentially modulate the degree of Kv1.3 tyrosine phosphorylation, the channel's biophysical properties, and the physical complexes associated with Kv1.3 in the presence of Src kinase.