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J. Biol. Chem., Vol. 280, Issue 17, 16772-16783, April 29, 2005

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Cell Signaling through the Protein Kinases cAMP-dependent Protein Kinase, Protein Kinase C, and RAF-1 Regulates Amphotropic Murine Leukemia Virus Envelope Protein-induced Syncytium Formation^*

Wei Wang, Zsolt Jobbagy, Terry H. Bird¶, Maribeth V. Eiden||, and Wayne B. Anderson**

From the Laboratory of Cellular Oncology, NCI, National Institutes of Health and the ^||Laboratory of Cellular and Molecular Regulation, NIMH, National Institutes of Health, Bethesda, Maryland 20892

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Cat siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKC, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKC (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Received for publication, October 12, 2004 , and in revised form, February 18, 2005.

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

Present address: Dept. of Pathology, Dartmouth Hitchcock Medical Center, One Medical Center Dr., Lebanon, NH 03756.

^¶ Present address: Dept. of Biology, Loyola College, 4501 N. Charles St., Baltimore, MD 21210-2699.

^** To whom correspondence should be addressed: Laboratory of Cellular Oncology, NCI, National Institutes of Health, Bldg. 37, Rm. 4124, 37 Convent Dr., Bethesda, MD 20892. Tel.: 301-496-9247; Fax: 301-480-0471; E-mail: andersow{at}mail.nih.gov.

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