N-TERMINAL c-FOS TYROSINE PHOSPHORYLATION REGULATES c-FOS/ER ASSOCIATION AND c-FOS-DEPENDENT PHOSPHOLIPID SYNTHESIS ACTIVATION

GABRIEL ORLANDO FERRERO; BEATRIZ LEONOR CAPUTTO

Rosario

Exposición; Jornadas de Lipidos 2008; 2008

We have found that c-Fos, a well known transcription factors, associates to the endoplasmic reticulum (ER) and activates phospholipid synthesis during cell growth and differentiation. 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).