Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

The relationship between SNPs in the genes of TLR signal transduction pathway downstream elements and rheumatoid arthritis susceptibility.

Toll-like receptors (TLRs) play an important role in the induction and regulation of the innate immune system or adaptive immune responses. Genetic variations within human TLRs have been reported to be associated with rheumatoid arthritis (RA). This study was conducted to investigate correlation between SNP of downstream mononucleotide in signal transduction of Toll-like receptors and predisposing genes of RA. There was obviously correlative between single nucleotide polymorphism and predisposing genes of RA. G-type of IL-1RAP rs766442 may be protecting genes of RA, while T-type alleles of IL-6R rs11265618 and IL-1RAP rs766442 may be susceptible genes of RA. In conclusion, the studies on the nucleis acid polymorphism in TLRs signal pathway contribute to disclose genes' influence on the attack mechanism of RA, early diagnosis and treatment of RA.

Role of the CDC25 homology domain of phospholipase Cepsilon in amplification of Rap1-dependent signaling.

Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC characterized by possession of CDC25 homology and Ras/Rap1-associating domains. We and others have shown that human PLCepsilon is translocated from the cytoplasm to the plasma membrane and activated by direct association with Ras at its Ras/Rap1-associating domain. In addition, translocation to the perinuclear region was induced upon association with Rap1.GTP. However, the function of the CDC25 homology domain remains to be clarified. Here we show that the CDC25 homology domain of PLCepsilon functions as a guanine nucleotide exchange factor for Rap1 but not for any other Ras family GTPases examined including Rap2 and Ha-Ras. Consistent with this, coexpression of full-length PLCepsilon or its N-terminal fragment carrying the CDC25 homology domain causes an increase of the intracellular level of Rap1.GTP. Concurrently, stimulation of the downstream kinases B-Raf and extracellular signal-regulated kinase is observed, whereas the intracellular level of Ras.GTP and Raf-1 kinase activity are unaffected. In wild-type Rap1-overexpressing cells, epidermal growth factor induces translocation of PLCepsilon to the perinuclear compartments such as the Golgi apparatus, which is sustained for at least 20 min. In contrast, PLCepsilon lacking the CDC25 domain translocates to the perinuclear compartments only transiently. Further, the formation of Rap1.GTP upon epidermal growth factor stimulation exhibits a prolonged time course in cells expressing full-length PLCepsilon compared with those expressing PLCepsilon lacking the CDC25 homology domain. These results suggest a pivotal role of the CDC25 homology domain in amplifying Rap1-dependent signal transduction, including the activation of PLCepsilon itself, at specific subcellular locations such as the Golgi apparatus.

RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation.

We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1.GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1DeltaRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1DeltaRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1DeltaRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1DeltaRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase.

Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras.

Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.

Role of Raf-1 conserved region 2 in regulation of Ras-dependent Raf-1 activation.

Full activation of Raf-1 requires the interaction of its CRD with Ras. The serine/threonine-rich region, CR2, of Raf-1 was implicated in Raf-1 regulation, but the underlying mechanism was unclear. Here we show that CRD loses its Ras-binding activity when expressed in connection with CR2, suggesting that CR2 masks CRD. This masking effect is abolished by substitution of Asp or Ala for Ser-259, a growth factor- and TPA-induced phosphorylation site in CR2. Treatment of COS-7 cells expressing Ha-Ras(Val-12) and Raf-1 with TPA enhances the Ha-Ras(Val-12)-dependent Raf-1 kinase activity. In contrast, the Ha-Ras(Val-12)-dependent activities of the Raf-1(S259D) and Raf-1(S259A) mutants are comparable to that of wild-type Raf-1 stimulated by both Ha-Ras(Val-12) and TPA and cannot be further stimulated by TPA treatment. These results suggest that the in vivo phosphorylation of Ser-259 may comprise a crucial step for Ras-dependent Raf-1 activation by unmasking CRD and promoting its association with Ras.

H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells.

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.

The strength of interaction at the Raf cysteine-rich domain is a critical determinant of response of Raf to Ras family small GTPases.

To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.

Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation.

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152-184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

Selective inhibition of Ras interaction with its particular effector by synthetic peptides corresponding to the Ras effector region.

Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.

Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS.

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.

Identification of PLC210, a Caenorhabditis elegans phospholipase C, as a putative effector of Ras.

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.

Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector.

Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.

Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal region interferes with ras-dependent activation of Raf-1.

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.

Membrane recruitment of Raf-1 is not the only function of Ras in Raf-1 activation.

Ras interacts with Raf-1 and stimulates its kinase activity, which results in activation of the mitogen-activated protein (MAP) kinase cascade. It has been proposed that the main function of Ras in Raf-1 activation is to recruit Raf-1 to the plasma membrane, where a separate activation event such as phosphorylation takes place. Here, we examined the activities of various mutants of human Ha-Ras to induce membrane translocation of Raf-1 and to activate Raf-1 in vivo. Overexpression of an activator region mutant Ha-Ras(V45E) in COS7 cells induced membrane translocation of Raf-1 as effectively as wild-type Ha-Ras. However, the activity of this mutant to activate Raf-1 and extracellular signal-regulated kinase-2 (ERK2) was attenuated by approximately 70% compared to that of wild-type Ha-Ras. The decrease in the specific activity was further demonstrated by measuring the activity of the Ha-Ras(V45E)-associated Raf-1 purified from the membrane fraction. These results imply that the association of Ras with Raf-1 has another important consequence, presumably dependent on the interaction between its activator region and Raf-1, than the simple recruitment of Raf-1 to the plasma membrane.

Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.

The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.