Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Leucine Carboxyl Methyltransferase

  • Scott L. Melideo
  • Jun Yong Ha
  • Jeffry B. Stock
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101594

Synonyms

Historical Background

Leucine carboxyl methyltransferase-1 and leucine carboxyl methyltransferase-2 (LCMT-1 and LCMT-2) are part of the methyltransferase superfamily (Petrossian and Clarke 2011). These enzymes transfer a methyl group from S-adenosylmethionine (SAM) to methyl-accepting substrates to produce the corresponding methylated product plus S-adenosylhomocysteine (SAH). Over 200 methyltransferase enzymes have been identified in the human genome, and almost a third of these have been linked to one or more different diseases. LCMT-1 (a.k.a. PPMT) was discovered by Lee and Stock (Lee and Stock 1993) as a protein phosphatase 2A (PP2A)-specific carboxyl methyltransferase (Fig. 1a). Posttranslational carboxyl methylation of PP2A plays a central role in the regulatory biochemistry of virtually all eukaryotic cells. Members of the PP2A phosphatase family that are carboxyl methylated by LCMT-1 include PP2A, PP4, and PP6 (Hwang et al. 2016). These enzymes account for over half of the total phosphoprotein phosphatase activity in most eukaryotic cells (Strack et al. 1997). Alterations in protein phosphatase activity associated with deficiencies in LCMT-1 have significant effects on every aspect of cellular physiology. LCMT-1 knockout mice exhibit an early embryonic lethal phenotype (Lee and Pallas 2007). This is not surprising, given that over a quarter of the proteins in a typical cell are subject to regulation by phosphorylation at one or more serine or threonine residues.
Leucine Carboxyl Methyltransferase, Fig. 1

(a) LCMT-1’s carboxyl methylation of Leu309 on PP2Ac. LCMT-1 catalyzes the addition of a methyl group on Leu309 (gray box) at the c-terminal of the catalytic subunit of PP2Ac. The rest of the subunit is represented as the R group shown in red. (b) LCMT-2’s production of wybutosine. Wybutosine is located at position 37 of tRNAphe. LCMT-2 catalyzes the addition of methyl and methoxycarbonyl groups (gray boxes) onto 7-(α-amino- α-carboxypropyl) wyosine forming wybutosine (Scheme was modified from Suzuki et al. (2009))

The majority of PP2A phosphatase activity is associated with PP2A holoenzyme heterotrimers composed of the highly conserved PP2A catalytic C subunit associated with a conserved scaffold-like A subunit and one of several different regulatory B subunits. Methylation of the alpha-carboxyl Leu309 at the carboxyl terminus of C subunits within AC dimers controls the subsequent association of different B subunits. This action in turn controls holoenzyme assembly and phosphoprotein substrate specificity (Tolstykh et al. 2000; Wu et al. 2000; Yu et al. 2001; Xing et al. 2008; Stanevich et al. 2011). Phosphatase carboxyl methylation is a reversible posttranslational modification. A specific methylesterase, PME, catalyzes the hydrolysis of the methyl ester formed by LCMT-1 to restore the phosphatase to its unmodified state with concomitant production of methanol (Ogris et al. 1999).

Although LCMT-2 is homologous to LCMT-1, it does not appear to be involved in protein phosphatase regulation (De Baere et al. 1999). Instead, the carboxyl methyl-accepting substrate of LCMT-2 is A37 in tRNAPhe. LCMT-2 is required for the final reactions in the biosynthesis of the unusual nucleotide base, wybutosine, at the A37 position in the anticodon loop of eukaryotic phenylalanine tRNA. LCMT-2 appears to catalyze the addition of both a methyl group and a methoxycarbonyl group (Fig. 1b) (Suzuki et al. 2009).

Gene and Structure of LCMT-1 and LCMT-2

LCMT-1 and LCMT-2 differ at both the genetic and structural level. LCMT-1, located at position 12.1 on chromosome 16 in the human genome, has 12 exons and two splice variants (A and B) (RefSeg mRNA). Isoform A, the main isoform, contains all 12 exons and isoform B is missing two (Fig. 2). LCMT-1 is a single-domain protein (Fig. 3). The LCMT-1 gene is found in all known eukaryotic genomes, and the active site residues Gly98, Asp122, and Glu198 in humans appear to be highly conserved in all eukaryotic species (Fig. 4).
Leucine Carboxyl Methyltransferase, Fig. 2

Alignment of LCMT-1 isoforms A and B. The uppercase letters are isoform A and the lowercase letters are isoform B

Leucine Carboxyl Methyltransferase, Fig. 3

Domain structure of LCMT-1 and LCMT-2. This green rectangle is the location of the methyltransferase domain (MT) that is common to the S-adenosylmethionine (SAM)-dependent methyltransferase superfamily. The domain is from amino acid 44–217 for LCMT-1 (a) and from residue 26–197 for LCMT-2 (b). The black square corresponds to the kelch domain (KD), which ranges from amino acid 494–540

Leucine Carboxyl Methyltransferase, Fig. 4

Alignment with human LCMT-1 and seven LCMT-1 orthologs. A Clustal Omega alignment of 8 known amino acid sequences for LCMT-1. The sequences are yeast (Saccharomyces cerevisae), damara mole-rat (Fukomys damarensis), bovine (Bos taurus), human (Homo sapien, highlighted in yellow), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), mouse (Mus musuculus), and rat (Rattus norvegicus). Conserved residues at the active site of LCMT-1 across species are in black boxes (human sequence residues Gly98, Asp122, and Glu198)

In contrast to LCMT-1, the LCMT-2 gene contains only one exon and has no known splice variants. LCMT-2 is composed of two domains: an N-terminal region that is homologous over its entire length to LCMT-1 and a C-terminal domain of approximately 250 amino acid residues that is unrelated to LCMT-1 and presumably functions to bind tRNAphe (Figs. 3a, b and 5) (Suzuki et al. 2009). The C-terminal domain of LCMT-2 is part of the Kelch-repeat superfamily. Kelch domains are scaffold-like structures composed of β-propeller repeats (Adams et al. 2000). LCMT-2 is found in many but not all eukaryotic species. An alignment of full-length human LCMT-2 and five other LCMT-2s showed four conserved cofactor binding site residues and one that was conserved with all of them expect in yeast (human residue Phe115 versus yeast Tyr147) (Fig. 6).
Leucine Carboxyl Methyltransferase, Fig. 5

Alignment of yeast LCMT-1 (PPM1) and LCMT-2 (TWY4)’s methyltransferase domain. (a) The sequence alignment of methyltransferase domain in LCMT-1 and LCMT-2. The alignment was carried out by clustal omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). (b) Structural alignment of LCMT-1 and LCMT-2. LCMT-1 is colored in orange (PDB code 1rjd) and LCMT-2 (PDB code 2zw9) is in green. The bound cofactor, S-adenosylmethionine (SAM), is represented in sticks. (c) Cofactor SAM binding sites of LCMT-1 and LCMT-2. The bound cofactors and their interacting residues are shown in sticks. The hydrogen bonds between SAM and LCMT-1 or LCMT-2 are represented in dotted lines. The residues showing differences in SAM binding are labeled in red

Leucine Carboxyl Methyltransferase, Fig. 6

Alignment with human LCMT-2 and five LCMT-2 orthologs. A Cobalt alignment of 6 known amino acid sequences for LCMT-2. The sequences are yeast (Saccharomyces cerevisae), hamster (Cricetulus griseus), fruit bat (Pteropus alecto), human (Homo sapiens, bolded), mouse (Mus musuculus), and rat (Rattus norvegicus). Conserved residues for cofactor binding across species are in black boxes (human sequence residues Arg59, Asp114, Asp158, and Tyr161)

LCMT-1 in Cancer

The PP2A phosphoprotein phosphatase system plays an important role in cellular growth regulation. Mutations that effect PP2A regulation are commonly associated with cancer. In a recent compilation of cancer genomic data sets, 73 different LCMT-1 mutations were identified most of which would be expected to interfere with PP2A methyltransferase activity (Fig. 7; Stanevich et al. 2011 and www.cbioprtal.org). These mutations include 64 missense mutations and 9 truncating mutations. LCMT-1 mutations are represented in various cancer types including lung, bladder, and cervical cancers (Table 1).
Leucine Carboxyl Methyltransferase, Fig. 7

Mutation sites of LCMT-1 in primary (a) and tertiary structure (b). Green and black circles (A) or spheres (B) represent missense and truncation mutations, respectively

Leucine Carboxyl Methyltransferase, Table 1

Mutations of LCMT-1 and their corresponding cancer

AA change

Mutation type

Cancer type

S9C

Missense

Lung squamous cell carcinoma

D20Y

Missense

Cervical squamous cell carcinoma

V26A

Missense

Stomach adenocarcinoma

L35M

Missense

Invasive breast carcinoma

A40V

Missense

Prostate adenocarcinoma

Y45H

Missense

Colorectal adenocarcinoma

A63T

Missense

Bladder urothelial carcinoma

G69V

Missense

Colorectal adenocarcinoma

R73Q

Missense

Glioblastoma multiforme

Non-small cell lung cancer

G76V

Missense

Non-small cell lung cancer

V77Sfs*5

FS del

Bladder urothelial carcinoma

A83T

Missense

Esophageal adenocarcinoma

R86G

Missense

Adrenocortical carcinoma

G98V

Missense

Non-small cell lung cancer

A99T

Missense

Pancreatic adenocarcinoma

G100S

Missense

Lung adenocarcinoma

F105L

Missense

Bladder urothelial carcinoma

L114F

Missense

Cutaneous melanoma

P124S

Missense

Cutaneous melanoma

E146*

Nonsense

Cervical squamous cell carcinoma

Q154E

Missense

Renal clear cell carcinoma

S162P

Missense

Uterine carcinosarcoma/uterine malignant mixed mullerian tumor

L172F

Missense

Cervical squamous cell carcinoma

R173*

Nonsense

Cutaneous melanoma

M187I

Missense

Endometrial carcinoma

P192S

Missense

Colorectal adenocarcinoma

E198Q

Missense

Lung adenocarcinoma

P206A

Missense

Lung adenocarcinoma

A210T

Missense

Diffuse large B-cell lymphoma

Papillary thyroid cancer

L212I

Missense

Cutaneous melanoma

L213Q

Missense

Head and neck squamous cell carcinoma

F225L

Missense

Invasive breast carcinoma

Papillary thyroid cancer

M241I

Missense

Non-small cell lung cancer

R246L

Missense

Colorectal adenocarcinoma

G272E

Missense

Colorectal adenocarcinoma

S277L

Missense

Head and neck squamous cell carcinoma

V279I

Missense

Endometrial carcinoma

D280N

Missense

Prostate adenocarcinoma

Uterine carcinosarcoma/uterine malignant mixed mullerian tumor

R287W

Missense

Lung adenocarcinoma

X295_splice

Splice

Papillary thyroid cancer

E297G

Missense

Diffuse large B-cell lymphoma

E304K

Missense

Cervical squamous cell carcinoma

G324*

Nonsense

Lung squamous cell carcinoma

G328W

Missense

Pancreatic adenocarcinoma

X328_splice

Splice

Non-small cell lung cancer

LCMT-1 in Neurodegenerative Diseases

Postmortem studies indicate that the levels of LCMT-1 expression in brains from individuals who suffered from Alzheimer’s (Sontag et al. 2004) are reduced to about half the levels found in brains from individuals who did not suffer from neurodegenerative disease. These reductions were associated with corresponding deficiencies in levels of PP2A carboxyl methylation (Sontag et al. 2004). In brain, carboxyl methylation of the PP2A AC heterodimeric catalytic core facilitates the binding of the B55alpha subunit to form the PP2A ACB55alpha heterotrimer, which is the form of PP2A in neurons that is primarily responsible for the dephosphorylation of tau (Bennecib et al. 2000; Kuszczyk et al. 2009; Martin et al. 2009) and alpha-synuclein (Lee et al. 2011). Hyperphosphorylation of tau and alpha-synuclein is thought to play a role in the etiology of Alzheimer’s and Parkinson’s disease, respectively. Unlike in cancer, where mutations in LCMT-1 are associated with disease, to date no genetic connections between LCMT-1 mutational variants and neurodegenerative disease have been established. Metabolic deficiencies in one-carbon methylation metabolism that lead to elevated total plasma homocysteine levels have been shown to be significant risk factors for idiopathic Alzheimer’s and Parkinson’s disease, and evidence suggests that this relates to deficiencies in PP2A carboxyl methylation (Vafai and Stock 2002; Sontag et al. 2008). For a detailed review explaining LCMT-1’s role in realtion to PP2A in Alzheimer’s disease, see Sontag and Sontag (2014).

Summary

LCMT-1 and LCMT-2 are homologous enzymes that carboxyl methylate different carboxylic acid substrates. In both cases, the methyl donor is S-adenosylmethionine. LCMT-1 specifically methylates the alpha carboxyl of the highly conserved C-terminal leucine residue in the PP2A catalytic subunit, and LCMT-2 methylates a tRNAPhe nucleotide. Deficiencies in LCMT-1 have been associated with numerous different cancers as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

Notes

Acknowledgments

We would like to thank Audrey Potts and David J. McFall for their help in putting together this manuscript. JBS has a significant financial interest in Signum Biosciences, a company devoted to the discovery of therapeutics that target the PP2A carboxyl methylation system.

References

  1. Adams J, Kelso R, Cooley L. The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol. 2000;10:17–24.PubMedCrossRefGoogle Scholar
  2. Bennecib M, Gong C-X, Grundke-Iqbal I, Iqbal K. Role of protein phosphatase-2A and-1 in the regulation of GSK-3, cdk5 and cdc2 and the phosphorylation of tau in rat forebrain. FEBS Lett. 2000;485:87–93.PubMedCrossRefGoogle Scholar
  3. De Baere I, Derua R, Janssens V, Van Hoof C, Waelkens E, Merlevede W, et al. Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue. Biochemistry. 1999;38:16539–47.PubMedCrossRefGoogle Scholar
  4. Hwang J, Lee JA, Pallas DC. Leucine carboxyl methyltransferase 1 (LCMT-1) methylates protein phosphatase 4 (PP4) and protein phosphatase 6 (PP6) and differentially regulates the stable formation of different PP4 holoenzymes. J Biol Chem. 2016;291:21008–19 : jbc. M116. 739920.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Kuszczyk M, Gordon-Krajcer W, Lazarewicz JW. Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau. Neurochem Int. 2009;55:174–80.PubMedCrossRefGoogle Scholar
  6. Lee J, Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem. 1993;268:19192–5.Google Scholar
  7. Lee JA, Pallas DC. Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells. J Biol Chem. 2007;282:30974–84.PubMedPubMedCentralCrossRefGoogle Scholar
  8. Lee K-W, Chen W, Junn E, Im J-Y, Grosso H, Sonsalla PK, et al. Enhanced phosphatase activity attenuates α-synucleinopathy in a mouse model. J Neurosci. 2011;31:6963–71.PubMedPubMedCentralCrossRefGoogle Scholar
  9. Martin L, Magnaudeix A, Esclaire F, Yardin C, Terro F. Inhibition of glycogen synthase kinase-3β downregulates total tau proteins in cultured neurons and its reversal by the blockade of protein phosphatase-2A. Brain Res. 2009;1252:66–75.PubMedCrossRefGoogle Scholar
  10. Ogris E, Du X, Nelson KC, Mak EK, Yu XX, Lane WS, et al. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A. J Biol Chem. 1999;274:14382–91.PubMedPubMedCentralCrossRefGoogle Scholar
  11. Petrossian TC, Clarke SG. Uncovering the human methyltransferasome. Mol Cell Proteomics. 2011;10:M110. 000976. doi:10.1074/mcp.M110.0000976.Google Scholar
  12. Sontag E, Luangpirom A, Hladik C, Mudrak I, Ogris E, Speciale S, White CL 3rd. Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol. 2004;63:287–301.Google Scholar
  13. Sontag JM, Sontag E. Protein phosphatase 2A dysfunction in Alzheimer’s disease. Front Mol Neurosci. 2014;7:16.Google Scholar
  14. Sontag J-M, Nunbhakdi-Craig V, Montgomery L, Arning E, Bottiglieri T, Sontag E. Folate deficiency induces in vitro and mouse brain region-specific downregulation of leucine carboxyl methyltransferase-1 and protein phosphatase 2A Bα subunit expression that correlate with enhanced tau phosphorylation. J Neurosci. 2008;28:11477–87.PubMedPubMedCentralCrossRefGoogle Scholar
  15. Stanevich V, Jiang L, Satyshur KA, Li Y, Jeffrey PD, Li Z, et al. The structural basis for tight control of PP2A methylation and function by LCMT-1. Mol Cell. 2011;41:331–42.PubMedPubMedCentralCrossRefGoogle Scholar
  16. Strack S, Westphal RS, Colbran RJ, Ebner FF, Wadzinski BE. Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments. Mol Brain Res. 1997;49:15–28.PubMedCrossRefGoogle Scholar
  17. Suzuki Y, Noma A, Suzuki T, Ishitani R, Nureki O. Structural basis of tRNA modification with CO2 fixation and methylation by wybutosine synthesizing enzyme TYW4. Nucleic Acids Res. 2009;37:2910–25.PubMedPubMedCentralCrossRefGoogle Scholar
  18. Tolstykh T, Lee J, Vafai S, Stock JB. Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits. EMBO J. 2000;19:5682–91.PubMedPubMedCentralCrossRefGoogle Scholar
  19. Vafai SB, Stock JB. Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer’s Disease. FEBS Lett. 2002;518:1–4.PubMedCrossRefGoogle Scholar
  20. Wu J, Tolstykh T, Lee J, Boyd K, Stock JB, Broach JR. Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. EMBO J. 2000;19:5672–81.PubMedPubMedCentralCrossRefGoogle Scholar
  21. Xing Y, Li Z, Chen Y, Stock JB, Jeffrey PD, Shi Y. Structural mechanism of demethylation and inactivation of protein phosphatase 2A. Cell. 2008;133:154–63.PubMedCrossRefGoogle Scholar
  22. Yu XX, Du X, Moreno CS, Green RE, Ogris E, Feng Q, et al. Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Bα regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen. Mol Biol Cell. 2001;12:185–99.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Scott L. Melideo
    • 1
  • Jun Yong Ha
    • 1
  • Jeffry B. Stock
    • 1
  1. 1.Department of Molecular BiologyPrinceton UniversityPrincetonUSA