Long chain acyl CoA synthetase 4 (Acs14) is a key enzyme in steroidogenesis. It participates in steroid synthesis through of arachidonic acid release and Steroidogenic Acute Regulatory protein (StAR) induction. Acs14 prefers arachidonic acid as substrate and acts probably as a homodimer. In steroidogenic cells, it has been demonstrated that Acs14 is a high turnover protein located mainly in mitochondrial-associated membrane fraction (MAM) bound to other proteins and that it is newly synthesized by hormone stimulation. The synthesis of Acs14 constitutes an early step in steroidogenesis. In the steroid synthesis process, activation of kinases plays a very important role. For this reason, the aim of this work was to study Acs14 as a possible phosphoprotein and try to elucidate the role of its phosphorylation. We have determined for the first time that Acs14 is a phosphoprotein whose phosphorylation is hormone-dependent. We also demonstrated that Acs14 acts effectively as a dimer and that phosphorylation occurs after dimer formation. Studies in vitro demonstrated that Acs14 is a substrate of both PICA and PKC and its phosphorylation by these kinases regulates its activity. (C) 2012 Elsevier Inc. All rights reserved.