Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils

Although important for apoptosis, the mechanism of Bax regulation is poorly understood. This study demonstrates that phosphorylation of Ser(184) regulates Bax activity. The phosphorylation required phosphatidylinositol 3-kinase/Akt activation and appeared to be mediated by Akt itself. In the serine-phosphorylated form, Bax was detected in the cytoplasm, could not be immunoprecipitated with the activation-specific antibody 6A7, and promoted heterodimerization with Mcl-1, Bcl-x(L), and A1. Apoptotic neutrophils possessed reduced levels of serine-phosphorylated Bax correlating with an increase in activated Bax as well as an increase in the amount of Bax found translocated to the mitochondria. We suggest that Bax is regulated by phosphorylation of Ser(184) in an Akt-dependent manner and that phosphorylation inhibits Bax effects on the mitochondria by maintaining the protein in the cytoplasm, heterodimerized with antiapoptotic Bcl-2 family members.     

Evidence that dystroglycan is associated with dynamin and regulates endocytosis

Disruption of the dystroglycan gene in humans and mice leads to muscular dystrophies and nervous system defects including malformation of the brain and defective synaptic transmission. To identify proteins that interact with dystroglycan in the brain we have used immunoaffinity purification followed by mass spectrometry (LC/MS-MS) and found that the GTPase dynamin 1 is a novel dystroglycan-associated protein. The beta-dystroglycan-dynamin 1 complex also included alpha-dystroglycan and Grb2. Overlay assays indicated that dynamin interacts directly with dystroglycan, and immunodepletion showed that only a pool of dynamin is associated with dystroglycan. Dystroglycan was associated and colocalized immunohistochemically with dynamin 1 in the central nervous system in the outer plexiform layer of retina where photoreceptor terminals are found. Endocytosis in neurons is both constitutive, as in non-neural cells, and regulated by neural activity. To assess the function of dystroglycan in the former, we have assayed transferrin uptake in fibroblastic cells differentiated from embryonic stem cells null for both dystroglycan alleles. In wild-type cells, dystroglycan formed a complex with dynamin and codistributed with cortactin at membrane ruffles, which are organelles implicated in endocytosis. Dystroglycan-null cells had a significantly greater transferrin uptake, a process well known to require dynamin. Expression of dystroglycan in null cells by infection with an adenovirus containing dystroglycan reduced transferrin uptake to levels seen in wild-type embryonic stem cells. These data suggest that dystroglycan regulates endocytosis possibly as a result of its interaction with dynamin.     

TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the formation of hamartomas in a wide range of human tissues. Mutation in either the TSC1 or TSC2 tumour suppressor gene is responsible for both the familial and sporadic forms of this disease. TSC1 and TSC2 proteins form a physical and functional complex in vivo. Here, we show that TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. Our data indicate a molecular mechanism for TSC2 in insulin signalling, tumour suppressor functions and in the inhibition of cell growth.     

cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.     

Evidence that glutamine is involved in neutrophil function

Phosphate-dependent glutaminase (PDG) activity, a key enzyme of glutamine metabolism, was determined in neutrophils obtained from the intra-peritoneal cavity (PC) or bronchoalveolar space (BAS) after administration of 1 ml or 100 microl, respectively of saline, glycogen solution (1%) or lipopolysaccharide (LPS 0.1 mg (100 microl)(-1)). Neutrophils were obtained by lavage of both sites with 20 ml saline 24 h after the administration of the stimuli. Glycogen and LPS, depending on the site the cells were obtained from, differently modulated PDG activity. Cells from BAS stimulated by glycogen or LPS had raised PDG activity to 30.5 +/- 5.2 and 42.7 +/- 12.1 nmol min(-1) mg(-1) protein, respectively, when compared with saline (9.1 +/- 0.9 nmol min(-1) mg(-1) protein); mean +/- SEM. On the other hand, cells from PC showed different PDG activity: 52.0 +/- 12.6 nmol min(-1) mg(-1) for saline, 36.5 +/- 9.5 nmol min(-1) mg(-1) for glycogen, and 76.6 +/- 11.2 nmol min(-1) mg(-1) for LPS; mean +/- SEM. Therefore, PDG activity varies with the site from which neutrophils are obtained and the stimulus imposed. The effect of glutamine on nitric oxide (NO) and tumour necrosis factor (TNF) production by peritoneal neutrophils, obtained after glycogen administration, cultured in the presence of LPS (0.5 microg ml(-1)) was also examined. The addition of glutamine at concentrations varying from 2 to 20 mM did not markedly affect NO production. Glutamine alone at 2 mM did not modify the production of TNF but in the presence of LPS caused a significant decrease. So, glutamine may preserve the function of neutrophils during infections and injuries.     

Human SMAD4 is phosphorylated at Thr9 and Ser138 by interacting with NLK

A polymorphism in the promoter region of uncoupling protein 2 gene ?866 G/A has been associated with its expression levels, the risk of obesity, and metabolic abnormalities. We aimed to investigate the associations of uncoupling protein (UCP)2 gene variants with obesity and related traits. A total of 440 subjects, 200 obese, and 240 non-obese individuals were included in this case–control study. Hormone and glucose levels were estimated using standard protocols. Genotyping of UCP-2 gene polymorphism for all subjects was performed by the PCR–RFLP polymerase chain reaction (PCR) method. Higher Systolic blood pressure, Diastolic blood pressure, Waist to hip ratio, Leptin, Insulin, and blood glucose levels were observed in obese than non-obese (P < 0.05). The distributions of genotype (0.001) and allele (0.003) were significantly different between the non-obese and the obese groups. In the obese group, subjects with the A allele showed significant high insulin levels (<0.001) in comparison with A allele non-carriers. In conclusion, our results suggest that the ?866 AA genotype and A allele of the UCP2 gene is associated with obesity and A allele associated with hyperinsulinemia in obese subjects.     

Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.     

Norovirus RNA-dependent RNA polymerase is phosphorylated by an important survival kinase, Akt

Viruses commonly use host cell survival mechanisms to their own advantage. We show that Akt, an important signaling kinase involved in cell survival, phosphorylates the RNA-dependent RNA polymerase (RdRp) from norovirus, the major cause of gastroenteritis outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the more prevalent norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs at a residue (Thr33) located at the interface where the RdRp finger and thumb domains interact and decreases de novo activity of the polymerase. This finding provides fresh insights into virus-host cell interactions.     

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.     

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [³²P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with ³²P_i, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [γ-³²P]ATP.     

Tyrosine 311 is phosphorylated by c-Abl and promotes the apoptotic effect of PKCdelta in glioma cells

In this study we characterized the phosphorylation of tyrosine 311 and its role in the apoptotic function of PKCdelta in glioma cells. We found that c-Abl phosphorylated PKCdelta on tyrosine 311 in response to H2O2 and that this phosphorylation contributed to the apoptotic effect of H2O2. In contrast, Src, Lyn, and Yes were not involved in the phosphorylation of tyrosine 311 by H2O2. A phosphomimetic PKCdelta mutant, in which tyrosine 311 was mutated to glutamic acid (PKCdeltaY311E), induced a large degree of cell apoptosis. Overexpression of the PKCdeltaY311E mutant induced the phosphorylation of p38 and inhibition of p38 abolished the apoptotic effect of the PKCdelta mutant. These results suggest an important role of tyrosine 311 in the apoptotic function of PKCdelta and implicate c-Abl as the kinase that phosphorylates this tyrosine.     

Evidence that the chromogranin B fragment 368-417 extracted from a pheochromocytoma is phosphorylated

A rabbit antiserum was raised against a synthetic peptide corresponding to residues 403 to 417 of human chromogranin B. This peptide was chosen to match the potential C-terminal end of a putative proteolytic fragment of the protein located between dibasic doublets in positions 366-367 and in positions 418-419 of the precursor. A radioimmunoassay based on this antiserum was developed and used to detect the protein or a fragment thereof in a pheochromocytoma tumor extract. One fragment was purified to homogeneity by successive reverse-phase HPLC chromatographies. The N-terminal sequence established by automated Edman degradation, was N-Y-P-S-L-E-L-D-K-M-A-H-G-Y-G-E-E-S-E-E-E-R corresponding to the 368-389 sequence of human chromogranin B. Taking into account the specificity of the antiserum used for peptide identification and alignment with the precursor sequence, we deduced that the purified peptide was chromogranin B (368-417) and represented a new peptide generated by limited proteolysis of chromogranin B. Combining electrospray mass-spectrometry and enzymatic dephosphorylation, we demonstrated that this peptide was phosphorylated.     

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

Folate is related to phosphorylated neurofilament-H and P-tau (Ser396) in rat brain

Protein phosphatase PP2A dephosphorylates phosphorylated tau (P-tau) and neurofilaments (pNFs). PP2A is S-adenosylmethionine (SAM)-dependent and might thus link methylation with neurodegeneration. Low SAM and increased S-adenosylhomocysteine (SAH) can enhance the risk of dementia. We studied the effect of hyperhomocysteinemia on P-tau (Ser396), pNF-H (heavy chain), and PP2A-activity and level (the C subunit) in rat brain. Wistar rats (total n=55) were fed either on a standard, a homocystine 1.7% or a methionine 2.4%-rich diet for 5 months. P-tau was tested in 21 frontal cortex tissue slices using immuno-fluorescence. Concentrations of pNF-H and the activity and level of PP2A were measured in brain extracts. Concentrations of homocysteine, SAM and SAH strongly increased in plasma of rats on the modified diets. The diets caused lowering of plasma folate and vitamin B12 and a significant increase in P-tau (Ser396) in brain tissues but PP2A activity and level were unchanged. Plasma folate correlated to brain tissue PP2A activity (r=0.28), pNF-H (r=-0.30), and P-tau (Ser396) staining (r=-0.57) all p<0.05. Phosphorylation of brain functional proteins was related to folate. The effect of the diet on P-tau and pNF-H seemed not to be explained by a lower activity or protein level of PP2A. Folate might prove protective against multiple steps in the process of neurodegeneration.