Up-regulation of the Pit-2 Phosphate Transporter/Retrovirus Receptor by Protein Kinase C ε

Abstract

The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced sodium-dependent phosphate (Na/P i ) uptake. Studies now have been carried out to determine which type of Na/P i transporter (Pit-1 or Pit-2) is regulated by PKC and which PKC isotypes are involved in the up-regulation of Na/P i uptake by the Na/P i transporter/viral receptor. It was found that the activation of short term (2-min) Na/P i uptake by PMA is abolished when cells are infected with amphotropic murine retrovirus (binds Pit-2 receptor) but not with gibbon ape leukemia retrovirus (binds Pit-1 receptor), indicating that Pit-2 is the form of Na/P i transporter/viral receptor regulated by PKC. The PKC-mediated activation of Pit-2 was blocked by pretreating cells with the pan-PKC inhibitor bisindolylmaleimide but not with the conventional PKC isotype inhibitor Gö 6976, suggesting that a novel PKC isotype is required to regulate Pit-2. Overexpression of PKC⑀, but not of PKC␣, -␦, or -, was found to mimic the activation of Na/P i uptake. To further establish that PKC⑀ is involved in the regulation of Pit-2, cells were treated with PKC⑀-selective antisense oligonucleotides. Treatment with PKC⑀ antisense oligonucleotides decreased the PMA-induced activation of Na/P i uptake. These results indicate that PMAinduced stimulation of Na/P i uptake by Pit-2 is specifically mediated through activation of PKC⑀.

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

“…Likewise, treatment with the PKC inhibitors GF109202X and Gö6976, or with the PKC activator PMA, did not affect GALV envelope protein-induced cell-cell fusion. These results are in agreement with our earlier studies, which showed that P i uptake mediated by PiT2, but not by PiT1, is up-regulated with exposure of cells to PMA (17). Of interest is the finding that the protein kinase inhibitor Rottlerin significantly decreased GALV envelope protein-induced cell-cell fusion (Table V).…”

“…Stimulation of A-MuLV Envelope Protein-induced Syncytium Formation by PKC-In earlier studies, it was established that activation of PKC by treatment of cells with PMA increased PiT2-mediated P i uptake, and that this effect was mediated through PMA activation of the PKC⑀ isoform (17). Thus, studies were initiated to determine if PKC⑀ also was involved in mediating A-MuLV envelope-induced cell-cell fusion.…”

“…J. DeClue and D. Lowy (National Institutes of Health). NIH3T3/hamster PiT2.FLAG and NMU34m/hamster PiT2.FLAG cells expressing FLAG epitope-tagged hamster PiT2 were constructed using the p⑀MTH.EAR.FLAG construct described above, and were generated as described previously (17). The PKC␣-, PKC␤-, PKC␦-, and PKC⑀-overexpressing NIH3T3 cell lines were generated as described previously utilizing the ⑀MTH vector (17).…”

“…NIH3T3/hamster PiT2.FLAG and NMU34m/hamster PiT2.FLAG cells expressing FLAG epitope-tagged hamster PiT2 were constructed using the p⑀MTH.EAR.FLAG construct described above, and were generated as described previously (17). The PKC␣-, PKC␤-, PKC␦-, and PKC⑀-overexpressing NIH3T3 cell lines were generated as described previously utilizing the ⑀MTH vector (17). NIH3T3/DN-PKC⑀ cells overexpressing the kinase-inactive mutant of PKC⑀ were constructed by converting lysine 437 within the catalytic domain to an arginine and then cloned into the epitope-tagging vector p⑀MTH, as described previously (24).…”

“…In earlier studies we reported that activation of PKC by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced, whereas activation of PKA inhibited, Na ϩ /P i uptake (16). In more recent studies, it was established that PMA treatment of cells enhances Na ϩ /P i uptake via stimulation of PiT2, and that this effect is specifically mediated through PMA activation of PKC⑀ (17). However, it remains to be determined if changes in protein kinase activities also can modulate PiT2 viral receptor function.…”