JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1.

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1-3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.

A novel mouse model for genetic variation in 10-formyltetrahydrofolate synthetase exhibits disturbed purine synthesis with impacts on pregnancy and embryonic development.

Genetic variants in one-carbon folate metabolism have been identified as risk factors for disease because they may impair the production or use of one-carbon folates required for nucleotide synthesis and methylation. p.R653Q (1958G>A) is a single-nucleotide polymorphism (SNP) in the 10-formyltetrahydrofolate (formylTHF) synthetase domain of the trifunctional enzyme MTHFD1; this domain produces the formylTHF which is required for the de novo synthesis of purines. Approximately 20% of Caucasians are homozygous for the Q allele. MTHFD1 p.R653Q has been proposed as a risk factor for neural tube defects (NTDs), congenital heart defects (CHDs) and pregnancy losses. We have generated a novel mouse model in which the MTHFD1 synthetase activity is inactivated without affecting protein expression or the other activities of this enzyme. Complete loss of synthetase activity (Mthfd1S(-/-)) is incompatible with life; embryos die shortly after 10.5 days gestation, and are developmentally delayed or abnormal. The proportion of 10-formylTHF in the plasma and liver of Mthfd1S(+/-) mice is reduced (P < 0.05), and de novo purine synthesis is impaired in Mthfd1S(+/-) mouse embryonic fibroblasts (MEFs, P < 0.005). Female Mthfd1S(+/-) mice had decreased neutrophil counts (P < 0.05) during pregnancy and increased incidence of developmental defects in embryos (P = 0.052). These findings suggest that synthetase deficiency may lead to pregnancy complications through decreased purine synthesis and reduced cellular proliferation. Additional investigation of the impact of synthetase polymorphisms on human pregnancy is warranted.

ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A.

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway.

FDH (10-formyltetrahydrofolate dehydrogenase) is strongly downregulated in tumors while its elevation suppresses proliferation of cancer cells and induces p53-dependent apoptosis. We have previously shown that FDH induces phosphorylation of p53 at Ser6, which is a required step in the activation of apoptosis. In the present study, we report that FDH-induced p53 phosphorylation is carried out by JNK1 and JNK2 (c-Jun N-terminal kinases) working in concert. We have demonstrated that FDH induces phosphorylation of JNK1 and JNK2, while treatment of FDH-expressing cells with JNK inhibitor SP600125, as well as knockdown of JNK1 or JNK2 by siRNA, prevents phosphorylation of p53 at Ser6 and protects cells from apoptosis. Interestingly, the knockdown of JNK1 abolished phosphorylation of JNK2 in response to FDH, while knockdown of JNK2 did not prevent JNK1 phosphorylation. Pull-down assay with the p53-specific antibody has shown that JNK2, but not JNK1, is physically associated with p53. Our studies revealed a novel mechanism in which phosphorylation of JNK2 is mediated by JNK1 before phosphorylation of p53, and then p53 is directly phosphorylated by JNK2 at Ser6.

Transport of rat liver glycine N-methyltransferase into rat liver nuclei.

Rat liver cytosolic glycine N-methyltransferase (GNMT) catalyzes the S-adenosylmethionine-dependent methylation of glycine to sarcosine. It is comprised of four identical 292-amino acid residue subunits. Recently, evidence has been provided to show that GNMT is identical to the cytosolic receptor for benzo[a]pyrene, which induces cytochrome P450 1A1 gene expression. In the present study we show that chemical modification of purified rat liver GNMT with fluorescein isothiocyanate (FITC) resulted in dissociation of the tetrameric enzyme and was accompanied by loss of enzyme activity. Amino acid sequence analysis of the FITC-labeled peptides obtained by hydrolysis of the modified protein with Staphylococcus aureus V8 protease revealed that lysines 45, 89, 92, 96, 122, and 147 were modified. Lys-122 and Lys-147 were derivatized in tetrameric, dimeric, and monomeric forms of the enzyme. Lysines 45, 89, 92, and 96 were derivatized only in monomeric GNMT, suggesting that modification of these residues resulted in GNMT dissociation. The modified monomeric GNMT was quickly transported into isolated rat liver nuclei. This transport was specific for the GNMT monomer, since neither tetramer nor dimer was able to enter the nuclei. Bovine carbonic anhydrase, similar in size to the GNMT monomer, was labeled with FITC to a similar extent but was not transported into the nuclei. Disruption of the nuclei containing fluorescein-labeled GNMT and subsequent extraction of the nuclear lysate with both high and low salt buffers recovered FITC-GNMT only in the chromatin pellet. Our study supports the suggestion of an additional function for GNMT, probably connected with regulation of cytochrome P450 1A1 gene expression.

Organization of the transcortin-binding domain on placental plasma membranes.

Complex formation between transcortin (corticosteroid-binding globulin) and 20 kDa sialoglycoprotein from human syncytiotrophoblast plasma membranes (presumably a transcortin-recognizing subunit of the transcortin membrane receptor) was studied using FPLC and cross-linking with bifunctional reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of the purified 20 kDa sialoglycoprotein and transcortin resulted in formation of covalently linked complexes of 95 kDa and 140 kDa consisting of one transcortin molecule and either two or four molecules of the membrane sialoglycoprotein (the molecular mass of transcortin is 55 kDa). Additionally, cross-linking resulted in the appearance of a 43 kDa species which is the cross-linked dimer of the membrane protein. The dimer was also observed during chromatography on a Superose 12 column in the absence of DFDNB treatment. Treatment of intact syncytiotrophoblast membranes with DFDNB resulted in isolation of the transcortin binding protein dimer as the major portion of total pool of the protein. Formation of the transcortin complexes with two and four molecules of the membrane protein was also observed when the membranes were incubated with 125I-labeled transcortin and treated with DFDNB, but formation of the latter complexes predominated. The results obtained suggest that the recognizing and binding domain for transcortin in placental membranes is organized as dimers consisting of non-covalently linked sialoglycoprotein monomers of a 20 kDa each and that transcortin has two sites for interaction with this dimer. Apparently, binding of two dimers results in the formation of the functional form of the transcortin-receptor complex. The possible biological role of such a complex is discussed.

Interaction of sex hormone-binding globulin with plasma membranes from the rat epididymis and other tissues.

The binding of human sex hormone-binding globulin (hSHBG) to plasma membranes prepared from the adult rat epididymis and other potential target and non-target tissues was examined. Specific binding sites were detected in the epididymis, testis, prostate, skeletal muscle and liver. The first three organs exhibited a higher (KD approx. 0.1 nM; Bmax approx. 0.05-0.10 pmol/mg membrane protein, Site I) and a lower (KD approx. 5 nM; Bmax approx. 1.0-2.5 pmol/mg membrane protein, Site II) affinity binding site. Only Site I was detected in muscle membranes and only Site II was detected in membranes isolated from liver. Specific binding was not detectable in either spleen or brain. Regional distribution of hSHBG binding sites occurred in the epididymis. Both Site I and Site II were present in the proximal caput and distal cauda. The distal caput and proximal cauda contained only Site II; no specific binding was detected in the corpus. Binding of hSHBG to epididymal membranes was time- and temperature-dependent. The presence of Ca2+ did not affect binding. Non-liganded [125I]-labeled hSHBG can bind to both sites in epididymal membranes. The affinity of hSHBG for Site I increased 2-fold when it was complexed with 5 alpha-dihydrotestosterone, testosterone or estradiol. The hSHBG-androgen complex had little effect on Site II versus steroid-free SHBG. However, the affinity of the hSHBG-estradiol complex for these sites was increased 10-fold. Cortisol, which has a low affinity for hSHBG, did not influence its binding to either the higher or lower affinity membrane sites.

Binding of human sex hormone-binding globulin-androgen complexes to the placental syncytiotrophoblast membrane.

We have found that human SHBG complexed with androgens binds specifically to the plasma membrane of human placental syncytiotrophoblast. Apparent equilibrium association constants were 5.3.10(11) M-1 for SHBG-testosterone complex and 1.1.10(11) M-1 for SHBG-5 alpha-dihydrotestosterone. Devoid of steroid, SHBG did not bind to the membrane. This suggests that the specific membrane binding of SHBG-androgen complexes is a step of the mechanism of androgen action on syncytiotrophoblast.