Global loss of leucine carboxyl methyltransferase-1 causes severe defects in fetal liver hematopoiesis.

Leucine carboxyl methyltransferase-1 (LCMT-1) methylates the C-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In this study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down ∼30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and usually death by embryonic day 16.5. Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit+Lin-Sca1+ cells. The percent cycling cells and mitotic indices of WT and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, noncompetitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multilineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis.

Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.

Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes.

O-Mannosylation is required for the solubilization of heterologously expressed human beta-amyloid precursor protein in Saccharomyces cerevisiae.

In an attempt to express human beta-amyloid precursor protein (APP) in yeast, we fortuitously found that this protein is only O-glycosylated in yeast. APP was effectively expressed in yeast, processed by yeast alpha-secretases, members of the Yapsin family, to produce N-terminal (sAPPalpha) and C-terminal (CTFalpha) domains, when its signal sequence was replaced by that of the yeast alpha-mating factor. APP is known to acquire N- and O-glycosylation through the endoplasmic reticulum (ER) and the Golgi apparatus and is transported to the plasma membrane in mammalian cells. In spite of the presence of canonical N-glycosylation consensus sequences, APP was not N-glycosylated in the yeast system. Pulse-chase experiments demonstrated that APP received only O-mannosylation in yeast. Examination of yeast pmt mutants, which are defective in the initiation of O-mannosylation in the ER, revealed that Pmt4p is most responsible for the oligosaccharide modification of APP. Maturation of APP was slowed down and aggregated forms of APP were observed by sucrose density gradient fractionation of the Deltapmt4 mutant lysate. This caused decreased production of CTFalpha. We conclude that O-mannosylation is required for the solubilization of exogenously expressed human APP.

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: evidence for translation during spermiogenesis.

An antiserum prepared against the purified protein carboxyl methyltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosis/atrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4-5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.

Protein-carboxyl methylation in adrenal medullary cells.

1. The protein-carboxyl methylating system has been studied in adrenal medullary cells either using disrupted cell components or with intact cells. Whereas the enzyme protein-carboxyl methylase (PCM) is cytosolic, the majority of its substrates is on or within chromaffin granules. With intact granules, methylation of surface proteins results in solubilization of membrane proteins. 2. Membrane PCM substrates have been identified as two proteins with apparent molecular weights of 55,000 and 32,000. Among the substrates located inside the granules, the chromogranins are excellent substrates, while dopamine beta-hydroxylase is poorly methylated. 3. Under physiological conditions, stimulation of the splanchnic nerve results in an increase in adrenal medullary protein-methyl ester formation as well as in an augmented methanol production. With adrenal medullary cells in culture, carboxyl-methylated chromogranin A is detected in mature chromaffin granules between 3 and 6 hr after labeling. Methylated chromogranins are secreted concomitantly with catecholamines following cholinergic stimulation. 4. These data coupled with those of Chelsky et al. (J. Biol. Chem. 262:4303-4309, 1987) on lamin B suggest that PCM methylates residues other than D-aspartyl and L-isoaspartyl in proteins. They further suggest that methylation may occur on nascent peptide chains before they are injected into the rough endoplasmic reticulum.

Is Ca2+-calmodulin-dependent protein phosphorylation in rat brain modulated by carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.