Limited redundancy in phosphorylation of retinoblastoma tumor suppressor protein by cyclin-dependent kinases in acute lymphoblastic leukemia

Cyclin-dependent kinases (CDKs) successively phosphorylate the retinoblastoma protein (RB) at the restriction point in G1 phase. Hyperphosphorylation results in functional inactivation of RB, activation of the E2F transcriptional program, and entry of cells into S phase. RB unphosphorylated at serine 608 has growth suppressive activity. Phosphorylation of serines 608/612 inhibits binding of E2F-1 to RB. In Nalm-6 acute lymphoblastic leukemia extracts, serine 608 is phosphorylated by CDK4/6 complexes but not by CDK2. We reasoned that phosphorylation of serines 608/612 by redundant CDKs could accelerate phospho group formation and determined which G1 CDK contributes to serine 612 phosphorylation. Here, we report that CDK4 complexes from Nalm-6 extracts phosphorylated in vitro the CDK2-preferred serine 612, which was inhibited by p16INK4a, and fascaplysin. In contrast, serine 780 and serine 795 were efficiently phosphorylated by CDK4 but not by CDK2. The data suggest that the redundancy in phosphorylation of RB by CDK2 and CDK4 in Nalm-6 extracts is limited. Serine 612 phosphorylation by CDK4 also occurred in extracts of childhood acute lymphoblastic leukemia cells but not in extracts of mobilized CD34+ hemopoietic progenitor cells. This phenomenon could contribute to the commitment of childhood acute lymphocytic leukemia cells to proliferate and explain their refractoriness to differentiation-inducing agents.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta1) and 22 kDa (beta2), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta1 and beta2 subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between beta1 and beta2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the alpha and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against alphaIIbbeta3 inhibited the aggregation response to stejnulxin, indicating that activation of alphaIIbbeta3 and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha2beta1 as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLC gamma 2. Mucetin-induced phosphorylation of the Fc gamma chain of platelet was greatly promoted by inhibition of alpha(IIb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of alpha(IIb)beta(3) activation are involved in dephosphorylation of Fc gamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(IIb)beta(3). Inhibition of alpha(IIb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A2 are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

Cytokine activation and disease progression in patients with stable moderate chronic heart failure

BACKGROUND: Activation of the cytokine and the complement system is associated with disease progression in severe congestive heart failure (CHF). Magnitude and prognostic relevance of cytokine and complement activation remain uncertain in patients with moderate CHF. OBJECTIVES: Measurement of cytokine and complement activation in patients with moderate CHF and testing whether C-reactive protein (CRP) can serve as a surrogate marker of their activation, adding independent prognostic information when co-measured with B-type natriuretic peptide (BNP). METHODS: The 118 study participants were separated into three groups based on pre-determined CRP and BNP levels: Group I (n = 27; CRP > 5 mg/liter, BNP > or = 200 pg/ml); Group II (n = 46; CRP < or = 5 mg/liter, BNP > or = 200 pg/ml); and Group III (n = 45; CRP < or = 5 mg/liter, BNP < 200 pg/ml). RESULTS: Mortality was high in Group I (30%; log-rank p < 0.001) but low in Groups II and III (2% and 4%, respectively; log rank, p = 0.7). No differences were observed for left ventricular ejection fraction (LVEF) and left ventricular end-diastolic diameter (LVEDD) between Groups I and II (31 +/- 16 vs 32 +/- 14% and 66 +/- 16 vs 65 +/- 11 mm, respectively), whereas in Group III LVEF was higher (42 +/- 17%, p = 0.002) with smaller LVEDD (57 +/- 13 mm, p = 0.012). Cytokine sCD14 and tumor necrosis factor (TNF)-alpha levels were not different between the three groups. However, interleukin-6 levels (9.75 +/- 8.17 pg/ml, p = 0.001) and the terminal complement complex C5b-9 (109.9 +/- 68 ng/ml; p = 0.04) were elevated in Group I, both correlating with CRP (interleukin-6: r = 0.5, p < 0.001; C5b-9: r = 0.41, p = 0.001). CONCLUSIONS: CRP may be used as a surrogate parameter for interleukin-6 and complement activation in moderate CHF. CRP in combination with BNP identifies a high-risk group with a tendency for poor outcome not discriminated by cardiac function.

Association of lipopolysaccharide-binding protein and coronary artery disease in men

OBJECTIVES: In this study we tested the hypothesis that lipopolysaccharide-binding protein (LBP) might be able to be used as a biomarker for coronary artery disease (CAD). BACKGROUND: The mechanisms by which the innate immune recognition of pathogens could lead to atherosclerosis remain unclear. Lipopolysaccharide-binding protein is the first protein to encounter lipopolysaccharide and to deliver it to its cellular targets, toll-like receptors; therefore, its presence might be a reliable biomarker that indicates activation of innate immune responses. METHODS: A total of 247 men undergoing elective coronary angiography were studied, and the extent of coronary atherosclerosis was assessed by 2 established scores: "extent score" and "severity score." Levels of LBP, markers of inflammation, and traditional risk factors for CAD were assessed. RESULTS: Serum LBP concentration was significantly increased in 172 patients with angiographically confirmed CAD compared with 75 individuals without coronary atherosclerosis (20.6 +/- 8.7 pg/ml vs. 17.1 +/- 6.0 pg/ml, respectively; p = 0.002). Moreover in multivariable logistic regression analyses, adjusted for established cardiovascular risk factors and markers of systemic inflammation, LBP was a significant and independent predictor of prevalent CAD (p < 0.05 in all models). CONCLUSIONS: Lipopolysaccharide-binding protein might serve as a novel marker for CAD in men. The present results underlie the potential importance of innate immune mechanisms for CAD. Further studies are warranted to bolster the data and to identify pathogenetic links between innate immune system activation and atherosclerosis.

Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein

Pleckstrin is a modular platelet protein consisting of N- and C-terminal pleckstrin homology (PH) domains, a central dishevelled egl10 and pleckstrin (DEP) domain and a phosphorylation region. Following agonist-induced platelet stimulation, dimeric pleckstrin translocates to the plasma membrane, is phosphorylated and then monomerizes. A recent study found that pleckstrin null platelets from a knockout mouse have a defect in granule secretion, actin polymerization and aggregation. However, the mechanism of pleckstrin signaling for this function is unknown. Our recent studies have led to the identification of a novel pleckstrin-binding protein, serum deprivation response protein (SDPR), by co-immunoprecipitation, GST-pulldowns and nanospray quadruple time of flight mass spectrometry. We show that this interaction occurs directly through N-terminal sequences of pleckstrin. Both pleckstrin and SDPR are phosphorylated by protein kinase C (PKC), but the interaction between pleckstrin and SDPR was shown to be independent of PKC inhibition or activation. These results suggest that SDPR may facilitate the translocation of nonphosphorylated pleckstrin to the plasma membrane in conjunction with phosphoinositides that bind to the C-terminal PH domain. After binding of pleckstrin to the plasma membrane, its phosphorylation by PKC exerts downstream effects on platelet aggregation/secretion.