N-terminal c-Fos tyrosine phosphorylation regulates c-Fos/ER association and c-Fos-dependent phospholipid synthesis activation

GABRIEL ORLANDO FERRERO; MAXIMILIANO MOISES PORTAL; BEATRIZ LEONOR CAPUTTO

Carlos Paz

Congreso; Reunión de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2008

Reunión de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

We have found that c-Fos, a well known transcription factors, associates to the endoplasmic reticulum (ER) and activates phospholipid synthesis. In T98G cells, c-Fos/ER association and consequently phospholipid synthesis activation is regulated by the phosphorylated state of c-Fos tyrosine residues. The small amount of c-Fos present in quiescent T98G cells is tyrosine-phosphorylated, is not associated to the ER and does not activate phospholipid synthesis. Furthermore, impairing tyr-dephosphorylation abrogates phospholipid synthesis activation and reduces proliferation rates to those of quiescent cells. c-Fos contains 4 phosphorylatable tyrosine residues at positions 10, 30, 106 and 337. Of these, only tyr 10 and 30 are relevant for this regulatory phenomenon. (Oncogene, 2007 26:3551-8). Objectives: We deemed of interest to determine which tyrosine-kinase(s) and tyrosine-phosphatase(s) are responsible for maintaining the phosphorylated or dephosphorylated state of c-Fos. Methods: Initially, an in silico search was performed using the Protein Human Data Base to find putative kinases or phosphatases acting on c-Fos. c-Src and EGFR were evidenced as putative kinases and TC-PTP phosphatase, respectively. To examine if c-Src phosphorylates tyr residues, recombinant c-Fos and c-Fos with tyr 10 and 30 substituted by phenylalanine were purified and incubated in vitro with recombinant c-Src. To determine if TC-PTP 45 dephosphorylates c-Fos, recombinant phosphatase was purified and incubated with phosphorylated-c-Fos (P-c-Fos). Results and Conclusions: c-Src specifically phosphorylates tyr residues 10 and 30 of c-Fos, while TC-PTP 45 removes both phosphates. When measuring phospholipid synthesis activation by c-Fos, or by double mutated c-Fos or by c-Fos-P, it was found that only dephosphorylated c-Fos, double mutated or not, associates to the ER membranes and activates phospholipid synthesis. We are currently studying if this is the mechanism that operates in vivo.