Nishit K. Mukhopadhyay

Tyrosine Phosphorylation of the Proto-Oncoprotein Raf-1 Is Regulated by Raf-1 Itself and the Phosphatase Cdc25A

ABSTRACT There is a growing body of evidence demonstrating that Raf-1 is phosphorylated on tyrosines upon stimulation of a variety of receptors. Although detection of Raf-1 tyrosine phosphorylation has remained elusive, genetic analyses have demonstrated it to be important for Raf-1 activation. Here we report new findings which indicate that Raf-1 tyrosine phosphorylation is regulated in vivo. In both a mammalian and baculovirus expression system, a kinase-inactive allele of Raf-1 was found to be tyrosine phosphorylated at levels much greater than that of wild-type Raf-1. The level of tyrosine phosphate on Raf-1 was markedly increased upon treatment with phosphatase inhibitors either before or after cell lysis. Cdc25A was found to dephosphorylate Raf-1 on tyrosines that resulted in a significant decrease in Raf-1 kinase activity. In NIH 3T3 cells, coexpression of wild-type Raf-1 and phosphatase-inactive Cdc25A led to a marked increase in Raf-1 tyrosine phosphorylation in response to platelet-derived growth factor. These data suggest that the tyrosine phosphorylation of Raf-1 is regulated not only by itself but also by Cdc25A.

Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis

BackgroundCholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry.ResultsDRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles.ConclusionWe describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events

Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu‐1, binds to collagen type IV (Coll IV) through β1‐integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450‐7). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu‐1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu‐1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu‐1 cells with 60 μM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu‐1 cell cytoskeleton integrity by 1–5 μM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19–6.6×10‐7) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation, but activated the ERKs in a dose dependent manner. β1‐integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/β1‐integrin mediated signaling events in Calu‐1 cells, which involve FAK, ERKs, and PKC.

MST1 Is a Multifunctional Caspase-Independent Inhibitor of Androgenic Signaling

The MST1 serine-threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR-chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa.

Histone deacetylation is directly involved in desilencing the expression of the catalytic subunit of telomerase in normal lung fibroblast

The regulation of telomerase expression in normal cells is poorly understood. Moreover, the molecular mechanism underlying tumor‐specific expression of telomerase remains unclear. We investigated the link between histone deacetylation and telomerase activity in normal lung and lung tumor cells. Four non‐small‐cell lung cancer (NSCLC) lines and one normal lung fibroblast line were tested for telomerase activity with or without Trichostatin A (TSA). The telomerase activity and the expression of telomerase associated components were determined by TRAP assay, RT‐PCR analysis and Western blot analysis. All 4 NSCLC cell lines exposed to 1μM TSA for 24h had no change in telomerase activity or hTERT mRNA level. Telomerase activity was very low in normal lung fibroblasts (mrc‐9) until exposed to 1μM TSA for 24h, at which time telomerase activity was readily detectable, with concomitant upregulation of hTERT mRNA (10‐fold). The level of other telomerase associated components (hTER and TP1) were unaltered. Furthermore, 1μM TSA exposure for 24h did not alter the level of c‐Myc or p21 mRNA. Immunodetection reveled that hTERT protein expression increased (∼6 fold) compared to c‐Myc, p21, or gelsolin. The effect of TSA on hTERT expression is independent of DNA methylation as judged by 5‐azacytidine (5aza) treatment. TSA effect on mrc‐9 cells is unaltered even in the presence of 200μg/ml cyclohexamide, suggesting a direct inhibition of histone deacetylation. Collectively, our study indicates that inhibition of histone deacetylation selectively regulates the transcriptional derepression of telomerase catalytic subunit in normal lung fibroblast cells compared to lung tumor cells.

Scaffold attachment factor B1 regulates the androgen receptor in concert with the growth inhibitory kinase MST1 and the methyltransferase EZH2

The androgen receptor (AR) is a transcription factor that employs many diverse interactions with coregulatory proteins in normal physiology and in prostate cancer (PCa). The AR mediates cellular responses in association with chromatin complexes and kinase cascades. Here we report that the nuclear matrix protein, scaffold attachment factor B1 (SAFB1), regulates AR activity and AR levels in a manner that suggests its involvement in PCa. SAFB1 mRNA expression was lower in PCa in comparison with normal prostate tissue in a majority of publicly available RNA expression data sets. SAFB1 protein levels were also reduced with disease progression in a cohort of human PCa that included metastatic tumors. SAFB1 bound to AR and was phosphorylated by the MST1 (Hippo homolog) serine-threonine kinase, previously shown to be an AR repressor, and MST1 localization to AR-dependent promoters was inhibited by SAFB1 depletion. Knockdown of SAFB1 in androgen-dependent LNCaP PCa cells increased AR and prostate-specific antigen (PSA) levels, stimulated growth of cultured cells and subcutaneous xenografts and promoted a more aggressive phenotype, consistent with a repressive AR regulatory function. SAFB1 formed a complex with the histone methyltransferase EZH2 at AR-interacting chromatin sites in association with other polycomb repressive complex 2 (PRC2) proteins. We conclude that SAFB1 acts as a novel AR co-regulator at gene loci where signals from the MST1/Hippo and EZH2 pathways converge.

The activation loop in Lck regulates oncogenic potential by inhibiting basal kinase activity and restricting substrate specificity.

The activities of Src-family non-receptor tyrosine kinases are regulated by structural changes that alter the orientation of key residues within the catalytic domain. In this study, we investigate the effects of activation loop mutations on regulation of the lymphocyte-specific kinase Lck (p56lck). Substitution of 5–7 residues amino terminal to the conserved activation loop tyrosine (Y394) increases kinase activity and oncogenic potential regardless of regulatory C-terminal tail phosphorylation levels (Y505), while most mutations in the 13 residues carboxyl to Y394 decrease kinase activity. Phosphorylation of the C-terminal regulatory tail is carried out by the cytosolic tyrosine kinase Csk and we find that mutations upstream or downstream of Y394 or mutation of Y394 do not affect the level of Y505 phosphorylation. In addition, we report that mutations on either side of Y394 affect substrate specificity in vivo. We conclude that the high degree of conservation across the entire activation loop of Src-family kinases is critical for normal regulation of kinase activity and oncogenicity as well as substrate selection.

Author Correction: Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

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HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A NOVEL REGULATOR OF ANDROGEN RECEPTOR TRANSLATION IN PROSTATE CANCER

Since the association of miR-125b with CaP is totally unknown, the purpose of this study was to understand the role of this miRNA in CaP. METHODS: LNCaP and cds1 cells cultured in androgendepleted medium were transfected with synthetic miR-125b and antimiR-125b, respectively, and cell proliferation was assessed using the WST-1 assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the loading of androgen receptor (AR) onto the 5’-DNA region of miR-125b. To test whether BAK1 mRNA is a direct target of miR-125b, a luciferase assay was performed in AR-negative DU145 cells to evaluate the effects of miR-125b on the 3’-untranslated region (3’-UTR) of the BAK1 gene. RESULTS: Synthetic miR-125b stimulated the growth of LNCaP cells in the absence of androgens. These cells retained typical morphological features. In contrast, in cds cells, inhibition of miR125b activity using synthetic anti-miR-125b inhibited proliferation of