J Biol Chem, Vol. 275, Issue 8, 5337-5346, February 25, 2000

Kv4.2 Phosphorylation by Cyclic AMP-dependent Protein Kinase* 

Anne E. Anderson§, J. Paige Adams¶, Yan Qian¶, Richard G. Cook||, Paul J. 
Pfaffinger¶, and J. David Sweatt¶ 

§From the Departments of  Pediatrics and Neurology, ¶ Division of Neuroscience,  
||Department of Microbiology and Immunology, Baylor College of Medicine, Houston, 
Texas 77030 

Recent evidence suggests that K+ channels composed of Kv4.2 -subunits underlie a 
transient current in hippocampal CA1 neurons and ventricular myocytes, and 
activation of the cAMP second messenger cascade has been shown to modulate this 
transient current. We determined if Kv4.2 -subunits were directly phosphorylated 
by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino 
and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion 
protein constructs; we observed that both of these fusion proteins were 
substrates for PKA in vitro. By using phosphopeptide mapping and amino acid 
sequencing, we identified PKA phosphorylation sites on the amino- and 
carboxyl-terminal fusion proteins corresponding to Thr38 and Ser552, 
respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA 
sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate 
PKA site phosphorylation in situ, phospho-selective antisera for each of the 
sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion 
protein, we observed that stimulation of the endogenous PKA cascade resulted in 
an increase in phosphorylation of Thr38 and Ser552 within Kv4.2 in the intact 
cell. We also observed modulation of PKA phosphorylation at these sites within 
Kv4.2 in hippocampal area CA1. These results provide insight into likely sites 
of regulation of Kv4.2 by PKA. 


* The costs of publication of this article were defrayed in part by the payment 
of page charges. The article must therefore be hereby marked "advertisement" in 
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 

§ To whom correspondence should be addressed: Cain Foundation Labs, BCM, Feigin 
Center MC 3-6365, 6621 Fannin St., Houston, TX 77030. Tel.: 713-798-3107; Fax: 
713-798-3946; E-mail: aander@cns.neusc.bcm.tmc.edu. 


Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, 
Inc. 




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