Kv4.2 is a locus for PKC and ERK/MAPK cross-talk.
Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.
Schrader
Laura A
LA
Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA. schrader@tulane.edu
Ren
Yajun
Y
Cheng
Feng
F
Bui
Dui
D
Sweatt
J David
JD
Anderson
Anne E
AE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D051672:Shal Potassium Channels / Q000378:metabolism*; D015398:Signal Transduction; D014981:Xenopus
D018076:DNA, Complementary; C496749:KCNIP3 protein, human; D051605:Kv Channel-Interacting Proteins; D012097:Repressor Proteins; D051672:Shal Potassium Channels; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases
Curr Opin Neurobiol. 2004 Jun;14(3):311-7
15194111
J Biol Chem. 2006 Apr 28;281(17):11769-79
16513649
FEBS Lett. 1991 Feb 25;279(2):256-60
1900473
Proteomics. 2004 Jun;4(6):1633-49
15174133
Am J Physiol Cell Physiol. 2006 Mar;290(3):C852-61
16251476
J Neurosci. 2006 Nov 22;26(47):12274-82
17122053
J Neurosci. 2002 Jun 15;22(12):4860-8
12077183
Circ Res. 1996 Oct;79(4):659-68
8831489
J Neurosci. 1999 Jun 1;19(11):4337-48
10341237
Trends Biochem Sci. 2000 Sep;25(9):448-53
10973059
Am J Physiol Heart Circ Physiol. 2001 Dec;281(6):H2518-27
11709419
Proc Natl Acad Sci U S A. 1988 Nov;85(22):8756-60
2903506
J Neurosci. 2000 Dec 1;20(23):8736-44
11102480
Neuron. 1994 Dec;13(6):1403-12
7993631
J Neurosci. 2002 Dec 1;22(23):10123-33
12451113
J Neurosci. 2006 Nov 22;26(47):12143-51
17122039
J Biol Chem. 2000 Feb 25;275(8):5337-46
10681507
Pflugers Arch. 1994 Mar;426(5):440-5
7517033
Am J Physiol. 1999 Jul;277(1):C100-10
10409113
Am J Physiol. 1998 Dec;275(6):H1963-70
9843794
J Biol Chem. 2003 Sep 19;278(38):36445-54
12829703
Neuron. 2006 Apr 6;50(1):89-100
16600858
Anal Biochem. 1992 May 15;203(1):173-9
1524213
Circ Res. 2005 Dec 9;97(12):1342-50
16293790
Nat Rev Neurosci. 2001 Jun;2(6):397-407
11389473
Physiol Rev. 1993 Apr;73(2):469-87
8386385
J Neurosci. 1998 May 15;18(10):3521-8
9570783
J Neurophysiol. 1999 Jan;81(1):408-11
9914302
Eur J Biochem. 1986 Nov 17;161(1):177-84
3023081
J Physiol. 1990 Dec;431:689-712
1983124
Am J Physiol. 1997 Oct;273(4):H1775-86
9362243
Genomics. 2000 Mar 1;64(2):144-54
10729221
J Neurochem. 2000 Dec;75(6):2277-87
11080179
J Physiol. 1990 Apr;423:257-77
1974922
Circ Res. 1994 Aug;75(2):252-60
8033339
Biochemistry. 1993 Feb 2;32(4):1032-9
8424932
J Neurophysiol. 2003 Sep;90(3):1671-9
12750419