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Differential Dephosphorylation of CTP: Phosphocholine

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Differential Dephosphorylation of CTP: Phosphocholine M BoC | ARTICLE Differential dephosphorylation of CTP:phosphocholine cytidylyltransferase upon translocation to nuclear membranes and lipid droplets Lambert Yuea,§, Michael J. McPheeb,§, Kevin Gonzaleza, Mark Charmanb, Jonghwa Leeb, Jordan Thompsonb, Dirk F. H. Winklerc, Rosemary B. Cornelld, Steven Pelecha,c, and Neale D. Ridgwayb,* aDepartment of Medicine, Division of Neurology, University of British Columbia, Vancouver, BC V6T 2B5, Canada; bDepartment of Pediatrics and Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada; cKinexus Bioinformatics Corporation, Vancouver, BC V6P 6T3, Canada; dDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada ABSTRACT CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate- Monitoring Editor limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α- James Olzmann helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of University of California, Berkeley the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we devel- oped phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the Received: Jan 8, 2020 P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the Revised: Mar 6, 2020 nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Accepted: Mar 10, 2020 Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephos- phorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addi- tion and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes. This article was published online ahead of print in MBoC in Press (http://www INTRODUCTION .molbiolcell.org/cgi/doi/10.1091/mbc.E20-01-0014) on March 11, 2020. Phosphatidylcholine (PC), the most abundant glycerophospholipid in S.P. is the president and chief scientific officer, as well as major and controlling mammalian cells, is synthesized de novo by the CDP-choline (Ken- shareholder in Kinexus Bioinformatics. nedy) pathway, by successive methylation of phosphatidylethanol- §L.Y. and M.M. made similar contributions and should both be considered as first authors. amine (PE) and by a salvage pathway involving acylation of lyso-PC. *Address correspondence to: Neale Ridgway ([email protected]). In most tissues, the CDP-choline pathway is the exclusive source of Abbreviations used: CCTα and PCYT1A, CTP:phosphocholine cytidylyltransfer- PC, but PE methylation provides a significant contribution to PC syn- ase-alpha; CCTβ and PCYT1B, CTP:phosphocholine cytidylyltransferase-beta; thesis in the liver (Vance and Ridgway, 1988) and for triglyceride stor- CDK1, cyclin-dependent kinase-1; CK2A, casein kinase 2A; DAG, diacylglycerol; ERK, extracellular-regulated kinase; HBSS, HEPES-buffered salt solution; LMNA/C, age in adipocytes (Horl et al., 2011). The CDP-choline pathway pro- lamin A/C; NE, nuclear envelope; nLD, nuclear lipid droplets; NLS, nuclear local- ceeds by the initial uptake of choline followed by its ATP-dependent ization signal; NR, nucleoplasmic reticulum; PKC, protein kinase C; PML, promy- elocytic leukemia; S-P, serine-proline; TBS-Tween, Tris-buffered saline-Tween. phosphorylation by choline kinases. CTP:phosphocholine cytidylyl- © 2020 Yue, McPhee, et al. This article is distributed by The American Society for transferase-alpha (CCTα) and CCTβ converts phosphocholine to Cell Biology under license from the author(s). Two months after publication it is avail- CDP-choline. Choline/ethanolamine phosphotransferases then utilize able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). CDP-choline and diacylglycerol (DAG) to form PC in the endoplasmic “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of reticulum. In most circumstances, CCT catalyzes the rate-limiting and the Cell®” are registered trademarks of The American Society for Cell Biology. regulated step in PC synthesis (Cornell and Ridgway, 2015). Volume 31 May 1, 2020 1047 FIGURE 1: Characterization of phosphosite-specific antibodies against P-domain phosphorylation sites in CCTα and CCTβ. (A) Alignment of human and rat CCTα and CCTβ2 sequences showing the location of phosphorylated serine (red) and tyrosine (green) residues, and the phosphopeptides (yellow boxes) used to generate antibodies against pS315+pS319 and pY359+pS362 phosphosites in human CCTα and CCTβ. Corresponding sequences for rat CCT are shown (green boxes). (B, C) Peptides with the amino acid sequences shown were synthesized on cellulose membranes using the SPOT-synthesis technique and each macroarray was probed with anti–CCTα/β-pS319 (B) or anti–CCTα- pY359+pS362 (C) as described in Materials and Methods. PCYT1A and PCYT1B encode homodimeric CCTα and CCTβ catalytically favorable conformation. M-domain interaction with PC- isoforms, respectively, that are composed of a conserved catalytic depleted membranes promotes its folding into a >60-residue am- domain followed by a membrane-binding amphipathic helix termed phipathic α-helix (Dunne et al., 1996; Taneva et al., 2003). M-domain the M-domain and a ∼50 residue C-terminal phosphorylation (P)- folding is promoted by electrostatic and hydrophobic interactions of domain (Figure 1A). The isoforms differ in their cellular location; an the N-terminal polybasic region and the C-terminal aromatic-en- N-terminal nuclear localization signal (NLS) in CCTα directs it to the riched segments, respectively (Arnold and Cornell, 1996; Johnson nucleus in most cells (Wang et al., 1995; Aitchison et al., 2015), while et al., 2003; Prevost et al., 2018). Engagement of CCT with cell CCTβ lacks an NLS and is cytoplasmic (Lykidis et al., 1998). The two membranes and its consequent activation thus depends on a lipid isoforms also slightly differ in the sequence of the P-domain composition that will effectively stabilize the α-helix conformation of (Figure 1A). Both CCTα and β are amphitropic enzymes that are in- the M-domain. active in their soluble forms but are activated by M-domain–medi- Membrane binding and α-helix stabilization of the CCTα M-do- ated translocation to membranes (Cornell and Ridgway, 2015). In main is antagonized by phosphorylation of the P-domain (Chong vitro, purified CCTα and CCTβ bind selectively to membrane vesi- et al., 2014).The CCT P-domain is highly phosphorylated; rat CCTα cles that are enriched in anionic lipids (e.g., fatty acids and phospha- contains 16 phosphoserine residues (MacDonald and Kent, 1994; tidic acid) or type II nonbilayer lipids (e.g., phosphatidylethanol- Cornell et al., 1995; Wang and Kent, 1995; Bogan et al., 2005), hu- amine and DAG; Cornell, 2016). Extensive structural studies of man CCTβ2 has 21 potential serine and threonine phosphosites soluble inactive CCTα show that an autoinhibitory helix of the M- (Dennis et al., 2011), and phosphoproteome analyses of human domain docks onto the αE helix positioned near the base of the liver have identified additional phosphothreonine and phosphoty- active site and interacts with catalytic residue K122, effectively com- rosine residues in CCTα and CCTβ (Bian et al., 2014). The P-domain peting for the substrate CTP (Huang et al., 2013; Lee et al., 2014; of soluble CCTα is highly phosphorylated relative to the active, Ramezanpour et al., 2018). Membrane binding by the M-domain membrane-associated enzyme and some residues are dephosphor- prevents interactions between the autoinhibitory helix and the ac- ylated upon membrane binding (Watkins and Kent, 1991; Wang tive site, freeing K122 and allowing the remodeling of αE into a et al., 1993; Houweling et al., 1994). Site-directed mutagenesis of 1048 | L. Yue, M. McPhee, et al. Molecular Biology of the Cell all 16 serine residues to glutamate in rat CCTα caused partial resis- peptides patterned after four of the most frequently detected and tance to membrane translocation in response to oleate, yet this evolutionarily conserved phosphorylated residues in human CCTα phosphomimetic mutant restored PC synthesis and the proliferation and CCTβ; S315+S319 and Y359+S362 (Supplemental Figure S1). of CCTα-deficient CHO cells, showing that the inhibitory effect of Using the SPOT technique of peptide synthesis on cellulose mem- P-domain phosphorylation is prevented by a strong lipid activator branes (Frank, 1992), macroarrays of peptides with systematic sub- (Wang and Kent, 1995). In vitro enzymatic analysis of P-domain de- stitutions at the targeted phosphosites were employed to establish letion and S315A mutants of CCTα indicated that phosphorylation the specificity of these antibodies. The antibody developed against caused negative cooperativity for activation by liposomes contain- the S315+S319 phosphorylation
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