-encoded choline kinase is an important regulatory enzyme that catalyzes the committed step in PC synthesis via the CDP-choline branch of the Kennedy pathway in S. cerevisiae
). Covalent modification by phosphorylation is an important mechanism by which the activity of an enzyme (and a metabolic pathway) may be regulated (46
). Previous studies have shown that the yeast choline kinase is phosphorylated and regulated by protein kinase A (48
). Phosphopeptide mapping analysis of choline kinase phosphorylated in vivo
and phosphorylated in vitro
by protein kinase A indicate that additional protein kinases are responsible for the phosphorylation of choline kinase (48
). In the present work, we addressed the hypothesis that choline kinase phosphorylation was also mediated by protein kinase C. In vitro,
protein kinase C phosphorylated choline kinase on serine residues resulting in the stimulation of activity. The phosphorylation of choline kinase was time- and dose-dependent, and dependent on the concentrations of choline kinase and ATP. These results indicated that the yeast choline kinase was a substrate for protein kinase C. To our knowledge, this is the first report of protein kinase C phosphorylation of a choline kinase from any organism.
Identification of the protein kinase C phosphorylation sites in choline kinase was addressed to gain information about the physiological relevance of this phosphorylation. Through a computer analysis, we identified five potential protein kinase C target sites in choline kinase (). Synthetic peptides with sequences for these sites were synthesized and examined for their ability to serve as substrates for protein kinase C. While all five peptides served as substrates, the peptides containing a protein kinase C phosphorylation motif at Ser25 and Ser37 were the best. This assay provided confidence that these residues might be major phosphorylation sites for protein kinase C. To provide support for this hypothesis, S25A and S37A mutations in choline kinase were constructed and expressed in cki1Δ eki1Δ cells. The S25A mutation had a major effect on the ability of choline kinase to be phosphorylated by protein kinase C. The extent of phosphorylation of this mutant was reduced by 60% when compared with the wild type enzyme. Moreover, phosphopeptide-mapping analysis of protein kinase C-phosphorylated choline kinase showed that two distinct phosphopeptides present in the wild type enzyme were greatly reduced in the S25A mutant enzyme. These data provided strong evidence that Ser25 was a specific target for protein kinase C phosphorylation. Extracts derived from cells expressing the S25A mutant exhibited reduced choline kinase activity. Thus, phosphorylation at Ser25 contributes to the activation of choline kinase activity. These results correlated with the stimulation of choline kinase activity upon protein kinase C phosphorylation in vitro.
The S37A mutant did not have a major effect on the protein kinase C phosphorylation of choline kinase. The phosphopeptide map of the S37A mutant was indistinguishable from that of the wild type enzyme, and the phosphopeptide map of the S25A, S37A mutant enzyme did not differ from that of the S25A mutant (data not shown). Accordingly, we did not consider Ser37 as a major protein kinase C phosphorylation site in choline kinase.
We addressed the question of whether the phosphorylation of choline kinase by protein kinase C affected the phosphorylation of the enzyme by protein kinase A, and vice versa
(i.e., hierarchal phosphorylation (83
)). Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. This effect could be attributed to a decrease in the rate of phosphorylation. An explanation for the reduced protein kinase C phosphorylation of the S30A mutant choline kinase was that Ser30
was a target site for protein kinases A and C. The choline kinase synthetic peptide containing Ser30
, which is a substrate for protein kinase A (49
), was also a substrate for protein kinase C. Moreover, a major phosphopeptide in the phosphopeptide map of the wild type protein kinase C-phosphorylated enzyme was absent in the phosphopeptide map of the S30A mutant enzyme. This same phosphopeptide results from the protein kinase A phosphorylation of choline kinase (49
). That one site is phosphorylated by multiple protein kinases is not uncommon (84
). For example, the yeast URA7
-encoded CTP synthetase is phosphorylated at Ser424
by both protein kinase A and protein kinase C (86
The choline kinase reaction in S. cerevisiae
is an important regulatory step in the synthesis of PC via the Kennedy pathway (17
). Accordingly, we questioned the physiological relevance of the protein kinase C phosphorylation of Ser25
with respect to PC synthesis in cells bearing the S25A and S25D mutant choline kinase enzymes. The in vivo
labeling studies showed that cells bearing the S25A mutation exhibited a decrease in PC synthesis whereas cells bearing the S25D mutation exhibited an increase in PC synthesis. These data supported the conclusion that activation of choline kinase activity by protein kinase C phosphorylation at Ser25
resulted in the stimulation of PC synthesis via the Kennedy pathway. However, the regulation of choline kinase activity by phosphorylation is complex. The enzyme is also phosphorylated by protein kinase A, and it may be phosphorylated by additional protein kinases. Thus, we cannot rule out other phosphorylation or regulatory events that might affect PC synthesis in cells carrying the S25A and S25D mutant choline kinase enzymes.
Previous studies have shown that the S30A (protein kinase A site) mutation in choline kinase results in a decrease in PC synthesis (49
). This observation has lead to the conclusion that protein kinase A phosphorylation at Ser30
stimulates PC synthesis via the Kennedy pathway (49
). As shown in the present work, the phosphorylation of choline kinase at Ser30
was also mediated by protein kinase C. Thus, the phosphorylation at Ser30
by both protein kinases should play a role in the regulation of PC synthesis via the Kennedy pathway. Additional studies will be required to determine the physiological conditions that protein kinase C phosphorylates choline kinase at Ser25
, and the conditions that protein kinase A phosphorylates the enzyme at Ser30
In mammalian cells, protein kinase C plays a central role in the transduction of lipid second messengers generated by receptor-mediated hydrolysis of membrane phospholipids (e.g., PC and phosphatidylinositol 4,5-bisphosphate) (88
). Phosphorylation of choline kinase by protein kinase C in S. cerevisiae
may represent a mechanism by which lipid signaling transduction pathways are coordinately regulated to PC synthesis and cell growth.