Phosphorylation of Saccharomyces cerevisiae Choline Kinase on Ser30 and Ser85 by Protein Kinase A Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway

Abstract

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1⌬ eki1⌬ double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of 3 H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser 30 and Ser 85 by protein kinase A regulates PC synthesis by the CDP-choline pathway.

Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana

“…Thus, it is probable that such regulation of the CKs depends on signaling pathways that differ according to the ionic or the osmotic nature of the stress (see [45] for a review). The discrepancy between the enhanced transcript levels and the unresponsiveness of the CK enzymes towards mannitol treatment raises the question of a possible phosphorylation-mediated post-translational regula- tion of the CKs, as observed in yeast [43], that would selectively affect the CK isoenzymes under hyperosmotic stress.…”

“…Indeed, stimulated CK activity is required for recycling Cho into phospholipase D (PLD)-mediated PC turnover signaling pathways in mammals [42]. In yeast, a change in the phosphorylation state of CK directly affects the pool of PC, demonstrating that post-translational modifications of CK regulates PC synthesis through the CDPCho pathway [43]. Since AtCK1 is inducible by wounding in Arabidopsis [44], our results denote that CKs also represent a potential regulatory target for some abiotic constraints in plants, such as increased environmental salinity.…”

CHKA (choline kinase alpha)

“…Although much work has been made in other organisms (Paddon et al 1982;Warden and Friedkin 1985;Kim and Carman 1999;Ramirez de Molina et al 2002;Yu et al 2002;Choi et al 2005;Soto 2008), little is known about human ChoKα regulation. It has been described that in HeLa cells, both EGF and insulin increase ChoK activity promoting the conversion of Cho to PCho, accompanied by an expansion of the PCho pool in treated cells (Uchida 1996).…”

Kinase joins the chaperone club: Androgen-regulated kinome reveals choline kinase alpha as a potential drug target in prostate cancer

“…The chaperone function of CHKA confers a growth advantage on PCa by stabilizing the AR in addition to its well-known function in fuelling membrane production through the Kennedy pathway 7 (Fig. 1), suggesting a de facto role for CHKA as a rate-limiting factor in cancer cell growth.…”