Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase

Membrane phosphatidylcholine homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP: phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane phosphatidylcholine content by reversible membrane binding and activation. Two prominent isoforms, CCT␣ and -␤ 2 , have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains but have divergent and structurally disordered N-terminal (N) and C-terminal phosphorylation (P) regions. We found that the binding affinity of purified CCT␤ 2 for anionic membranes was weaker than CCT␣ by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCT␣ can be attributed in large part to the electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured N-terminal segment of CCT␣ but lacking in CCT␤ 2 . The membrane partitioning of CCT␤ 2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCT␣ and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane binding motif not only in vitro but in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P-truncated forms showed that the in vitro membrane affinity of CCT␤ 2 is more sensitive than CCT␣ to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

Section: Discussionmentioning

confidence: 99%

“…The complete set of kinases responsible for the phosphorylation of the 16 sites in CCT␣ has not been identified (47,(51)(52)(53)(54), and the kinases other than Cdk5 (11) that act on CCT␤ 2 are unknown. In vitro SLV binding analysis suggested that region P truncation has a similar effect on membrane binding affinity as dephosphorylation.…”

mentioning

confidence: 99%

The Intrinsically Disordered Nuclear Localization Signal and Phosphorylation Segments Distinguish the Membrane Affinity of Two Cytidylyltransferase Isoforms

Dennis¹,

Taneva²,

Cornell

2011

“…9). Phosphorylation of CCT by ERK is known to reduce activity, rather than increase activity (50), so it is unlikely that CCT is a substrate for ERK in the context of differentiating neuronal cells.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylcholine Biosynthesis during Neuronal Differentiation and Its Role in Cell Fate Determination

Marcucci

Paoletti

Jackowski

et al. 2010

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase ␣ (CK␣) and choline cytidylyltransferase ␣ (CCT␣). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT␣. Later, the transcription of CK␣-and CCT␣-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK␣ or CCT␣ increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT␣ or CK␣ was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.

“…Serine-phosphorylated cortactin is localized in the sites of dynamic actin assembly in tumor cells (30). It is likely that as with many proline-directed kinases, ERK assembles within a scaffolding complex with multiple effectors at a docking domain or region within sites of cortactin (52). This assembly of kinases and regulatory scaffolding molecules might then provide a phosphodegron-like signature for recruitment of SCF…”

Section: Discussionmentioning

confidence: 99%

Extracellular Signal-regulated Kinase (ERK) Regulates Cortactin Ubiquitination and Degradation in Lung Epithelial Cells

Zhao

Wei

Mialki

et al. 2012

Self Cite

Background: Cortactin is essential for barrier integrity. Results: ERK inhibition attenuates cortactin phosphorylation, ubiquitination, and degradation via the ␤-Trcp-mediated ubiquitin-proteasome system. Conclusion: Cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network. Significance: Understanding cortactin stability provides new targets to regulate epithelial barrier integrity.