B-Raf Phosphorylation Research Articles

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

The mTOR pathway controls phosphorylation of BRAF at T401

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion.

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers. This study shows the anticancer mechanisms of the PCAIs in two breast cancer cell lines, MDA-MB-468 and MDA-MB-231. PCAIs (NSL-YHJ-2-27) treatment increased BRAF phosphorylation, whereas CRAF phosphorylation significantly decreased in both cell lines. Moreover, the PCAIs also stimulated the phosphorylation of MEK, ERK, and p90RSK by 116, 340, and 240% in MDA-MB-468 cells, respectively. However, in MDA-MB-231 cells, a significant increase of 105% was observed only in p90RSK phosphorylation. Opposing effects were observed for AKT phosphorylation, whereby an increase was detected in MDA-MB-468 cells and a decrease in MDA-MB-231 cells. The PCAIs also induced apoptosis, as observed in the increased pro-apoptotic protein BAK1, by 51%, after treatment. The proportion of live cells in PCAIs-treated spheroids decreased by 42 and 34% in MDA-MB-468 and MDA-MB-231 cells, respectively, which further explains the PCAIs-induced apoptosis. The movement of the cells through the Matrigel was also inhibited by 74% after PCAIs exposure, which could have been due to the depleted levels of F-actin and vinculin punctate, resulting in the shrinkage of the cells by 76%, thereby impeding cell movement. These results show promise for PCAIs as potential therapies for TNBC as they significantly inhibit the hallmark processes and pathways that promote cell proliferation, migration, and invasion, which result in poor prognoses for breast cancer patients.

WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells.

Multiple Wilms tumor gene 1 (WT1) splicing variants are expressed in mammals, and these variants regulate tumorigenesis and mediate the development of multiple tissues and organs, including gonads. However, WT1 splicing variants (+KTS or -KTS) are expressed in only two nonmammalian vertebrates, and unexpectedly, their functions in chicken ovaries remain elusive. Progesterone (P4) secreted by chicken granulosa cells (GCs) participates in various physiological processes and plays an important role in maintaining reproductive performance. The purpose of this study was to investigate the effect of WT1(+KTS) and WT1(-KTS) on chicken P4 secretion in preovulatory GCs. First, we detected WT1 mRNA expression in GCs from follicles of different developmental stages by Quantitative real-time PCR (RT-qPCR) and found that WT1 mRNA expression was considerably increased in preovulatory GCs compared with prehierarchical GCs. Primary cells collected from preovulatory follicles were treated with WT1(+KTS) or WT1(-KTS) overexpression vectors and subsequently cultured in the absence or presence of follicle-stimulating hormone (FSH). The mRNA levels of FSH-receptor (FSHR) and steroidogenesis genes were determined by RT-qPCR, and the P4 levels in the cell supernatants were measured by radioimmunoassay (RIA). Both WT1(+KTS) and WT1(-KTS) significantly decreased P4 secretion due to a reduction in FSHR, STAR and CYP11A1 mRNA levels. Western blotting revealed that ERK1/2 and BRAF phosphorylation levels were suppressed after overexpression of WT1(+KTS) or WT1(-KTS), whereas total protein and mRNA levels were not significantly changed. In addition, CREB protein and phosphorylation levels were inhibited after overexpression of WT1(+KTS) or WT1(-KTS). In conclusion, WT1(+KTS) and WT1(-KTS) inhibited CREB protein activity and significantly reduced FSHR, STAR and CYP11A1 mRNA levels, which subsequently suppressed FSH-induced P4 secretion in preovulatory GCs by modulating ERK1/2 signaling.

BRAF Exon 15 Mutations in Papillary Carcinoma and Adjacent Thyroid Parenchyma: A Search for the Early Molecular Events Associated with Tumor Development.

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples—Only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

Deep Small RNA Sequencing of BRAF V600E Mutated Papillary Thyroid Carcinoma With Lymph Node Metastasis.

Papillary thyroid carcinoma (PTC) is the most predominant subtype of thyroid cancer, contributing to more than 80% of all thyroid or endocrine malignancies. Its prognosis is relatively good compared to other cancers, with more than 90% overall 10-year survival rate (Haugen et al., 2016). However, despite the favorable prognosis, some cases exhibit aggressive phenotype. On average, 50% of the patients are presented with lymph node metastases (LNM) at diagnosis (Saiselet et al., 2015). LNM in PTC confers various poor prognostic indicators; it increases recurrence risk and decreases long term survival predominantly in patients older than 45 years old (Lundgren et al., 2006; Xing, 2013). Treating recurrent PTCs is still a challenge (Pasternak and Shen, 2017) and one of the important issues yet to be solved in PTC patient management is the mortality and morbidity associated with the recurrent disease (Lee et al., 2013). MicroRNAs (miRNAs) are short, endogenous noncoding RNAs first identified in Caenorhabditis elegans which regulate gene expression by binding to the 3′-UTR of target messenger RNA (mRNAs). Their post-transcriptional regulatory functions are involved in controlling the level of proteins involved in numerous biological processes, including embryogenesis, organogenesis, tissue homeostasis, immune system function and cell cycle control (Tafrihi and Hasheminasab, 2019). Relationship between miRNAs and tumor growth, tumor progression and metastasis has been demonstrated by many studies, indicating the ability of these molecules to be used as biomarkers for diagnosis and prognosis (Iorio and Croce, 2012). MiRNA also plays a role as biomarker in predicting lymph nodes metastasis (LNM) in thyroid cancer (Ab Mutalib et al., 2016; Mutalib et al., 2016; Mohamad Yusof et al., 2018). P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a quite recently discovered class of RNAs and more than 30,000 piRNA genes been identified within the human genome (Zhang et al., 2014; Wang et al., 2019). Originally described as key regulators for the germline maintenance and transposon silencing, these short RNAs were disregarded for a long time due to our limited knowledge regarding their function (Weng et al., 2019). However, emergent of new data reveals that unusual expression of piRNAs is a distinct feature Fin many diseases, including cancers (Weng et al., 2019). This has changed our perspectives on their significance in various diseases. Epigenetically, piRNAs are also the post-transcriptional regulators of the expression of specific downstream target genes (Watanabe and Lin, 2014), and accumulating evidence demonstrates that similarly to miRNAs, piRNAs also possess both oncogenic and tumor-suppressive roles (Weng et al., 2019). BRAF is a serine-threonine kinase that is activated by RAS binding and protein recruitment to the cell membrane. Activation of MEK along with the MAPK signaling pathway is activated by BRAF phosphorylation (Nikiforov and Nikiforova, 2011). The most frequent genetic changes in PTC are point mutations of BRAF which are observed in 35 to 70% of PTC cases (Li et al., 2012; Xing, 2013). More than 95% of BRAF mutations in thyroid cancers are thymine to adenine transversion at position 1799 (T1799A) which result in the substitution of glutamate from valine at residue 600 (V600E) (Xing et al., 2015). Various studies have also shown that the BRAF V600E mutation is related to LNM, as further presented by a meta-analysis by Song et al. (2018). In the past years, the evolution of next-generation sequencing technologies has enabled global transcriptome expression profiling and the discovery of novel human miRNAs (Kozomara and Griffiths-Jones, 2011; Kozomara and Griffiths-Jones, 2014; Kozomara et al., 2019) and piRNAs (Weng et al., 2019). Coincidentally, PTC has a relatively lower overall mutation burden hence termed as quiet genome (Cancer Genome Atlas Research Network, 2014; Siraj et al., 2016), therefore investigating the regulatory molecules such as the miRNAs and piRNAs has the potential to deepen our understanding of this cancer. This Data Report aims to provide the readers a comprehensive dataset of small RNAs expression derived from next-generation sequencing in an unbiased manner. For detailed analysis of this dataset focusing on miRNAs with biological insights in PTC, please refer to our original article published in Frontiers of Endocrinology (Mohamad Yusof et al., 2018).

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.

Abstract C260: Dual targeting of the PI3K/Akt/mTOR pathway significantly delays chondrosarcoma progression and relapse.

Abstract Chondrosarcoma is a rare and usually slow growing sarcoma. This tumor is notoriously resistant to chemotherapy and radiation treatment. To improve survival rates in patients with chondrosarcoma exploring targeted therapies approaches seems promising. Thus we performed a preclinical study evaluating the efficacy of mTOR inhibitor everlimus in an orthotopic rat chondrosarcoma model and showed the potential of this mTORC 1 inhibitor to significantly delay tumor progression and relapse. Based on these data, we initiated preclinical studies in the same chondrosarcoma model to investigate how mTOR inhibition can be optimized. We first evaluated the antitumor effect of a PI3K (GDC-0941), an Akt (G-594) and a dual PI3K/mTOR inhibitor (GDC-0980). In a second step and in order to overcome forms of resistance we combined inhibition of PI3K to Akt or PI3K to the one of mTOR. GDC-0941 based drug combinations (GDC-0941+G-594; GDC-0941+RAD001) potency to inhibit tumor progression was compared to the one of each agent alone. For each setting, the effect of each treatment on mTOR and ERK pathways, apoptosis and cell proliferation was also tested. Results: As monotherapy, PI3K inhibitor (GDC-0941) was found to be the most efficient inducing an inhibition of tumor progression of 58 % whereas RAD001 induced a 49 % inhibition. The comparison of therapeutic efficiency of GDC-0941 based combinations showed an additive effect of G-594 or RAD001 to GDC-0941. Each of these combination caused an inhibition >60% in chondrosarcoma progression, whereas each agent alone induced a 22% to 45 % inhibition of tumor progression. GDC-0941 exposure inhibited chondrosarcoma proliferation as evaluated by Ki67 staining and induced Caspase 3/7 mediated apoptosis when used as single agent or in combined therapies. Our data reveal that all combination treatments blocked Akt activation, and inhibited phosphorylation of both 4EBP1 and S6K1 which resulted in. The tested treatments induced a similar inhibition of mTOR mediated signaling. Chondrosarcoma exposure to GDC-0941 or G-594 caused a 20% inhibition of p70S6K and 4EBP activation similar to the one induced by the rapalog RAD001. The PI3K inhibitor GDC-0941 caused an activation of Erk Pathway as noted by the strong increase in bRaf phosphorylation. Taken together these data confirm that targeting the PI3K/mTOR pathway is an efficient treatment for chondrosarcoma and combined therapy best way to cause inhibition of tumor progression. The effect noted on Erk pathway suggests that new combination approaches targeting the mTOR and Erk pathway could be of interest. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C260. Citation Format: Delphine Jury, Anne Valerie Decouvelaere, Amandine Pasquier, Marcia Belvin, Lorie Friedman, Jean Baptiste Langlois, Jean Philippe Michot, Jean Yves Blay, Aurelie Dutour. Dual targeting of the PI3K/Akt/mTOR pathway significantly delays chondrosarcoma progression and relapse. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C260.

Braking BRAF: AMPK Leaves ERK Stranded in the Desert

In this issue, Shen etal. (2013) show that BRAF phosphorylation by AMPK attenuates BRAF signaling and inhibits cell proliferation. This identifies BRAF as a novel AMPK substrate and further reveals how these pathways interact.

AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (∆4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated B-Raf regulation was also detected in a panel of PCa lines derived from the cPten−/−L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells.

The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl‐2 genes in non‐small cell lung cancer

Polyomaviruses such as BK virus (BKV), JC virus (JCV) and Merkel cell polyomavirus (MCPyV) are typically nononcogenic, although they have been detected in a variety of human neoplasms. The aim of our study was to determine the frequency of the most common polyomaviruses MCPyV, BKV and JCV as well as the gene expression profile of genes involved in oncogenesis including K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in a cohort of non-small cell lung cancer (NSCLC) patients. Real-time and nested polymerase chain reaction (PCR) were used to assess the presence of polyomaviruses DNA in tissue biopsies from 110 patients with primary NSCLC and 14 tissue specimens from macroscopically healthy sites of their lung. Real-time PCR was also used to determine the mRNA expression of K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in selected samples. Results showed that ten NSCLC specimens were positive for the presence of MCPyV DNA (10/110, 9.1%), whereas no control sample was tested positive for the virus. The MCPyV-positive samples were predominantly obtained from male smokers (9/10). BKV and JCV DNA were not detected either in lung tissues biopsies or the control specimens. Interestingly, gene expression analysis revealed increased mRNA and protein expression of BRAF gene in association with BRAF phosphorylation in the MCPyV-positive samples, whereas Bcl-2 gene expression was downregulated in the same type of samples. The detected MCPyV prevalence in NSCLC in combination with the deregulated expression of BRAF and Bcl-2 genes suggests that these events are likely to contribute to the pathogenesis of NSCLC.

Simalikalactone E (SkE), a new weapon in the armamentarium of drugs targeting cancers that exhibit constitutive activation of the ERK pathway

Simalikalactone E (SkE) is a quassinoid extracted from a widely used Amazonian antimalarial remedy. Although SkE has previously been shown to have cytostatic and/or cytotoxic activities in some tumor cell lines, its mechanism of action has not yet been characterized. We show here that SkE in the high nanomolar range inhibited the growth of various leukemic and solid tumor cell lines. Importantly, SkE was highly efficient at inhibiting chronic myelogenous leukemia (CML) cells that exhibit constitutive activation of the MAPK pathway and, accordingly, it impaired the phosphorylation of ERK1/2. SkE also abrogated MEK1/2 and B-Raf phosphorylation but had no effect on Ras activity. Moreover, SkE was particularly effective against melanoma cell lines carrying the B-Raf-V600E mutation. Importantly, SkE resensitized the PLX-4032-resistant 451Lu melanoma cell line (451Lu-R) and was more efficient than U0126, a MEK inhibitor, and PLX-4032 (PLX) at inducing the apoptosis of two hairy cell leukemia (HCL) patient samples carrying the B-Raf-V600E mutation. Finally, SkE was as efficient as imatinib at inhibiting tumor formation in a xenograft model of CML cells in athymic mice. In conclusion, we show that SkE, a very potent inhibitor of B-Raf-V600E, is highly effective against cancer cell lines that exhibit constitutive activation of the ERK1/2 pathway.

P3‐036: Aß inhibition of kinesin 5 induces neurotransmitter and neurotrophin receptor mislocalization and dysfunction

second biosimulation test used a combination of 25 mM brain derived neurotrophic factor (BDNF) and 10 m M NGF as a continuous exposure for 60 min duration; this approach evaluated stimulation of p75 NTR, TrkA, and TrkB. Based on the human microarray results demonstrating downregulation of TrkA (50%) and TrkB (60%), the corresponding parameters in the TTR biosimulation were decreased by the same amount. Results: Baseline results were validated from published literature on neuronal calcium levels mediated via the phospholipase C-g and inositol-3-phosphate pathway at both bolus doses of NGF alone. With the corresponding parameters decreased in the TTR biosimulation, Figure 1: A) The reaction flux for c-RAF1 phosphorylation of MEK1 was delayed to peak value by 1.5 min from exposure, but the peak value was increased to 5 times the baseline value; B) Moreover, a slight shift to the right of the flux over time was observed with the B-RAF phosphorylation of MEK1. Conclusions: These biosimulation results indicate that the transient response to neurotrophins is accentuated, whereas the sustained MEK activating pathway is blunted in AD, and may be potential mechanisms for the failure of pro-survival pathways in AD.

The phosphorylation specificity of B-RAFWT, B-RAFD594V, B-RAFV600E and B-RAFK601E kinases: An in silico study

Phosphorylation of the B-RAF kinase is an essential process in tumour induction and maintenance in several cancers. Herein the phosphorylation specificity of the activation segment of the wild type B-RAF kinase and the B-RAF(D594V), B-RAF(V600E) and B-RAF(K601E) mutants was examined by molecular dynamics (MD) simulations and GRID molecular interaction field analysis. According to our analysis, Thr599 and Ser602 were the only residues in the activation segment in B-RAF(WT) that were well exposed to ATP binding, which is in agreement with the experimental results, and provide a molecular basis of the observed phosphorylation. The phosphorylation specificity was altered significantly for each of the three different mutants studied due to the large conformational changes and subsequent alterations in the electrostatic forces between several residues for each of these mutants. Thus the analysis revealed limited phosphorylation potential of the non-active B-RAF(D594V) mutant and several potential ATP binding sites were identified for the highly active B-RAF(V600E) mutant. The Lys601 residue, which is specific to RAF and not present in the activation segment of other similar kinases, was identified to potentially be of major importance to the observed differences in the phosphorylation specificity of the mutants. Our results indicate that Lys601 might be a specific ATP coordinating residue, contributing to the B-RAF phosphorylation specificity. The overall results can be helpful for the understanding of the B-RAF phosphorylation processes on a molecular level.

PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

Rheb Inhibits C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network.

Microarrays for the Functional Analysis of the Chemical-Kinase Interactome

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals for monitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC50 of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.

Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

B-Raf and Raf-1 Are Regulated by Distinct Autoregulatory Mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.