Transmembrane Gla Protein 4 as a Novel Modulator of ERK2.

Abstract Transmembrane Gla protein 4 (TMG4) is a single pass, type I transmembrane protein with a vitamin-K dependent γ-carboxy-glutamic acid-rich domain (Kulman et al PNAS,2001). Bioinformatic analysis of the primary sequence indicates a very high degree of conservation (ca. 80%) among mammals and to-date no known functional roles have been assigned to this protein. In our efforts to elucidate molecular functions of TMG4, we first analyzed sub cellular localization of this protein. Microscopic analysis of HEK293T cells expressing a TMG4-GFP fusion construct showed juxtanuclear localization of the tagged protein at 24h post transfection. Staining with organelle specific fluorescent dyes revealed that the TMG4-GFP primarily localizes to the trans Golgi network in the cell. Recently, there have been increasing number of reports of golgi-localized proteins participating in compartmentalized signal transduction pathways and affecting cell differentiation and proliferation by activating major signaling cascades like the Ras-Raf/MAPK signaling cascades. Moreover, the transmembrane localization and the current lack of evidence for a direct role for TMG4 in the coagulation pathways encouraged us to hypothesize that TMG4 may play a role in signal transduction. To test this we generated a TMG4 over-expressing cell line (high-TMG4) by stably transfecting HEK293T cells with a CMV-promoter driven TMG4 plasmid , and a TMG4 knock-down cell line (low-TMG4) by stable transfections with a TMG4 short-hairpin RNA (shRNA) expression vector. The levels of TMG4 gene expression were confirmed in both the cell lines by RNA and protein expression analysis. The low-TMG cells showed >90% knockdown of TMG4 in comparison to untransfected control cells. The cells were grown under standard tissue culture conditions for 48h after which they were harvested. Total cell lysates from the cell lines were analyzed by western blotting using primary antibodies against a panel of signal transduction proteins representing major signaling pathways including threonine kinase, tyrosine kinase and ERK2. The results showed no significant changes in activation of signal transduction proteins tested except for extracellular-signal-regulated kinase (ERK) -2. The high-TMG cells showed a down modulation of phosphorylated ERK2 while the low-TMG4 cell lysates showed a 500-fold up regulation of phosphorylated ERK2 when compared to the levels in the low-TMG cells. Our observations were confirmed visually by assaying the cells for intracellular localization of ERK2. We used immuno-fluorescent staining to show that TMG4 levels regulate ERK2 activation based on TMG4 dependent translocation of ERK2 using anti phospho-ERK2 and anti-ERK2 antibodies. More than 85% of high-TMG4 cells showed extra nuclear localization of ERK2 while phosphorylated ERK2 was localized in the nucleus in the >80% of low-TMG4 cells. Thus there is an inverse correlation between TMG4 levels and ERK2 phosphorylation. We conclude that TMG4 is a molecular modulator of ERK2. Whether TMG4 has a direct interaction or affects ERK2 indirectly is under investigation. These studies suggest novel roles for vitamin-K dependent Gla-proteins in regulating thrombopoietin dependent megakaryocytic differentiation, platelet activation and myelopoiesis and myeloproliferation.