pp60 -mediated Phosphorylation of Connexin 43, a Gap Junction Protein

doi:10.1074/jbc.270.21.12751

pp60 -mediated Phosphorylation of Connexin 43, a Gap Junction Protein (*)

From the (1) Molecular Carcinogenesis Program, Cancer Research Center of Hawaii and Department of Genetics and Molecular Biology, School of Medicine, and the
(2) Mathematics-Science Department, Kapiolani Community College, University of Hawaii at Manoa, Honolulu, Hawaii 96813

^¶ To whom correspondence should be addressed. E-mail: aflau{at}uhunix.uhcc.hawaii.edu.

↵^§ Present address: Molecular Biology and Virology Laboratory, Salk Institute, La Jolla, CA 92138.

Abstract

Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60 and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60 is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60 regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.

Footnotes

↵* This research was supported by Grant CA 52098 from the NCI, National Institutes of Health and Grant VM-21A from the American Cancer Society (to A. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GJC

gap junctional communication

Cx43

connexin 43

GST

glutathione S-transferase

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

MES

2-(N-morpholino)ethanesulfonic acid

mAb

monoclonal antibody

TEA

triethanolamine

RSV

Rous sarcoma virus.
↵²W. Kurata, unpublished observations.