N-terminal Kinase Domain Research Articles

Mechanisms underlying Plk1 polo-box domain-mediated biological processes and their physiological significance.

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key regulator of various cell cycle events that are critical for proper M-phase progression. The polo-box domain (PBD) present in Plk1’s C-terminal non-catalytic region has been shown to play a central role in targeting the N-terminal kinase domain of Plk1 to specific subcellular locations. Subsequent studies reveal that PBD binds to a phosphorylated motif generated by one of the two mechanisms - self-priming by Plk1 itself or non-self-priming by a Pro-directed kinase, such as Cdc2. Here, we comparatively review the differences in the biochemical steps of these mechanisms and discuss their physiological significance. Considering the diverse functions of Plk1 during the cell cycle, a better understanding of how the catalytic activity of Plk1 functions in concert with its cis-acting PBD and how this coordinated process is intricately regulated to promote Plk1 functions will be important for providing new insights into different mechanisms underlying various Plk1-mediated biological events that occur at the multiple stages of the cell cycle.

HIP1–ALK, a Novel Fusion Protein Identified in Lung Adenocarcinoma

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Dueling Kinases Regulate Cell Size at Division through the SAD Kinase Cdr2

Cell size control requires mechanisms that integrate cell growth and division. Key to this integration in fission yeast is the SAD family kinase Cdr2, which organizes a set of cortical nodes in the cell middle to promote mitotic entry through Wee1 and Cdk1. Cdr2 is inhibited by a spatial gradient of the DYRK kinase Pom1 emanating from cell tips in a cell-size-dependent manner, but how the Pom1 gradient inhibits Cdr2 activity during cell growth is unknown. Here, we show that Pom1 acts to prevent activation of Cdr2 kinase activity by the CaMKK Ssp1. We found that Ssp1 activates Cdr2 through phosphorylation of a conserved threonine residue (Thr166) in the activation loop of the Cdr2 N-terminal kinase domain both in vitro and in cells. The levels of this activating phosphorylation increased with cell-cycle progression, and genetic epistasis demonstrated that Ssp1 promotes mitotic entry through Cdr2. Intriguingly, Pom1 phosophorylated the C-terminal domain of Cdr2, and this modification reduced Cdr2-T166 phosphorylation by Ssp1. These findings show how activation of the conserved mitotic inducer Cdr2 is integrated with an inhibitory spatial gradient to ensure proper cell size control at mitosis.

Class III Myosin Motor Activity Correlates with Localization in Actin Protrusions

Class III myosins (myo3A and myo3B) contain a N-terminal kinase domain, central motor domain, and an isoform specific C-terminal tail. Myo3A contains a longer tail and has an actin-binding motif at the C-terminus that is not present in the shorter tail of myo3B. Modifications to the myo3A gene result in late onset human deafness (DFNB30), suggesting myo3B may be able to compensate for myo3A only early in life. Thus, it is critical to understand differences in the motor properties of myo3A and myo3B. We investigated the in vitro motility of myo3A and myo3B, each with the kinase domain removed and containing two IQ domains and a C-terminal GFP fusion (myo3AΔK.GFP and myo3BΔK.GFP). The sliding velocity measured in the in vitro motility assay was ∼20% faster in myo3AΔK.GFP (76 nm/sec) compared to myo3BΔK.GFP (62 nm/sec). The sliding velocity correlated well with the maximum actin-activated ATPase activity (kcat = 1.0 sec−1 and 0.7 sec−1, respectively). Interestingly, myo3B.ΔK.GFP contained a 25-fold weaker actin affinity compared to myo3A.ΔK.GFP as assessed by the actin-dependence of the ATPase activity (KATPase = 3 and 76 µM, respectively). We transfected N-terminally GFP tagged myo3A.ΔK containing the full length tail domain into COS7 cells and examined the efficiency of localizing to the filopodia tips by examining the tip to cell body ratio. Since the tail actin binding motif is required for localization to filopodia tips, we generated a chimera that contained the myo3B motor and the actin binding motif of the myo3A tail. The tip localization efficiency was approximately 17% (p<0.05) reduced in myo3B compared to myo3A. Thus myo3B is a slightly slower motor with a weaker actin affinity compared to myo3A, which may explain why it is unable to compensate for the loss of myo3A in DFNB30.

Classic phenotype of Coffin–lowry syndrome in a female with stimulus‐induced drop episodes and a genotype with preserved N‐terminal kinase domain

An adolescent female presented with intellectual disability, stimulus-induced drop episodes (SIDEs), facial characteristics that include wide set eyes, short nose with wide columella, full and everted lips with wide mouth and progressive skeletal changes: scoliosis, spondylolisthesis and pectus excavatum. These findings were suggestive of Coffin-Lowry syndrome (CLS), and this was confirmed by the identification of a novel mutation in RPS6KA3, a heterozygous one basepair duplication at nucleotide 1570 (c.1570dupA). This mutation occurs within the C-terminal kinase domain of the protein, and, therefore contradicts the previous report that SIDEs is only associated with premature truncation of the protein in the N-terminal kinase domain or upstream of this domain. As CLS is X-linked, it is unusual for a female to have such a classic phenotype.

The DAPK family: a structure–function analysis

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain. ZIPK's activity is independent of CaM but can be activated by DAPK. The three kinases share some common functions and substrates, such as induction of autophagy and phosphorylation of myosin regulatory light chain leading to membrane blebbing. Furthermore, all can function as tumor suppressors. However, they also each possess unique functions and intracellular localizations, which may arise from the divergence in structure in their respective C-termini. In this review we will introduce the DAPK family, and present a structure/function analysis for each individual member, and for the family as a whole. Emphasis will be placed on the various domains, and how they mediate interactions with additional proteins and/or regulation of kinase function.

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Crystal Structure of a Human IκB Kinase β Asymmetric Dimer

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.

Structural basis for the inhibition of Polo-like kinase 1

Polo-like kinase 1 (PLK1) is a master regulator of mitosis and is considered a potential drug target for cancer therapy. PLK1 is characterized by an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD). The KD and PBD are mutually inhibited, but the molecular mechanisms of the autoinhibition remain unclear. Here we report the 2.3-Å crystal structure of the complex of the Danio rerio KD and PBD together with a PBD-binding motif of Drosophila melanogaster microtubule-associated protein 205 (Map205(PBM)). The structure reveals that the PBD binds and rigidifies the hinge region of the KD in a distinct conformation from that of the phosphopeptide-bound PBD. This structure provides a framework for understanding the autoinhibitory mechanisms of PLK1 and also sheds light on the activation mechanisms of PLK1 by phosphorylation or phosphopeptide binding.

Identification of Arabidopsis VTC3 as a putative and unique dual function protein kinase::protein phosphatase involved in the regulation of the ascorbic acid pool in plants

Ascorbic acid (AsA) is present at high levels in plants and is a potent antioxidant and cellular reductant. The major plant AsA biosynthetic pathway is through the intermediates D-mannose and L-galactose. Although there is ample evidence that plants respond to fluctuating environmental conditions with changes in the pool size of AsA, it is unclear how this regulation occurs. The AsA-deficient Arabidopsis thaliana mutants vtc3-1 and vtc3-2 define a locus that has been identified by positional cloning as At2g40860. Confirmation of this identification was through the study of AsA-deficient At2g40860 insertion mutants and by transgenic complementation of the AsA deficiency in vtc3-1 and vtc3-2 with wild-type At2g40860 cDNA. The very unusual VTC3 gene is predicted to encode a novel polypeptide with an N-terminal protein kinase domain tethered covalently to a C-terminal protein phosphatase type 2C domain. Homologues of this gene exist only within the Viridiplantae/Chloroplastida and the gene may therefore have arisen along with the D-mannose/L-galactose AsA biosynthetic pathway. The vtc3 mutant plants are defective in the ability to elevate the AsA pool in response to light and heat, suggestive of an important role for VTC3 in the regulation of the AsA pool size.

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)-associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss-of-function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N-terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild-type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen-activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast-based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.

Inhibition of SypG-Induced Biofilms and Host Colonization by the Negative Regulator SypE in Vibrio fischeri

Vibrio fischeri produces a specific biofilm to promote colonization of its eukaryotic host, the squid Euprymna scolopes. Formation of this biofilm is induced by the sensor kinase RscS, which functions upstream of the response regulator SypG to regulate transcription of the symbiosis polysaccharide (syp) locus. Biofilm formation is also controlled by SypE, a multi-domain response regulator that consists of a central regulatory receiver (REC) domain flanked by an N-terminal serine kinase domain and a C-terminal serine phosphatase domain. SypE permits biofilm formation under rscS overexpression conditions, but inhibits biofilms induced by overexpression of sypG. We previously investigated the function of SypE in controlling biofilm formation induced by RscS. Here, we examined the molecular mechanism by which SypE naturally inhibits SypG-induced biofilms. We found that SypE’s N-terminal kinase domain was both required and sufficient to inhibit SypG-induced biofilms. This effect did not occur at the level of syp transcription. Instead, under sypG-overexpressing conditions, SypE inhibited biofilms by promoting the phosphorylation of another syp regulator, SypA, a putative anti-sigma factor antagonist. Inhibition by SypE of SypG-induced biofilm formation could be overcome by the expression of a non-phosphorylatable SypA mutant, indicating that SypE functions primarily if not exclusively to control SypA activity via phosphorylation. Finally, the presence of SypE was detrimental to colonization under sypG-overexpressing conditions, as cells deleted for sypE outcompeted wild-type cells for colonization when both strains overexpressed sypG. These results provide further evidence that biofilm formation is critical to symbiotic colonization, and support a model in which SypE naturally functions to restrict biofilm formation, and thus host colonization, to the appropriate environmental conditions.

Crystal Structure and Mechanism of Activation of TANK-Binding Kinase 1

Tank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 Å and 4.0 Å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation.

Regulatory motifs in Chk1

Chk1 is the effector kinase of the G2 DNA damage checkpoint. Chk1 homologs possess a highly conserved N-terminal kinase domain and a less conserved C-terminal regulatory domain. In response to DNA damage, Chk1 is recruited to mediator proteins assembled at lesions on replication protein A (RPA)-coated single-stranded DNA (ssDNA). Chk1 is then activated by phosphorylation on S345 in the C-terminal regulatory domain by the PI3 kinase-related kinases ATM and ATR to enforce a G2 cell cycle arrest to allow time for DNA repair. Models have emerged in which this C-terminal phosphorylation relieves auto-inhibitory regulation of the kinase domain by the regulatory domain. However, experiments in fission yeast have shown that deletion of this putative auto-inhibitory domain actually inactivates Chk1 function. We show here that Chk1 homologs possess a kinase-associated 1 (KA1) domain that possesses residues previously implicated in Chk1 auto-inhibition. In addition, all Chk1 homologs have a small and highly conserved C-terminal extension (CTE domain). In fission yeast, both of these motifs are essential for Chk1 activation through interaction with the mediator protein Crb2, the homolog of human 53BP1. Thus, through different intra- and intermolecular interactions, these motifs explain why the regulatory domain exerts both positive and negative control over Chk1 activation. Such motifs may provide alternative targets to the ATP-binding pocket on which to dock Chk1 inhibitors as anticancer therapeutics.

Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution.

Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45-346) of mouse RSK2, or RSK2(NTKD), has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3'',4''-di-O-acetyl-α-L-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-L-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2(NTKD) with a dissociation constant (K(d)) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K(d) for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca(2+).

Improving the Affinity of SL0101 for RSK Using Structure-Based Design

Enhanced activity of the Ser/Thr protein kinase, RSK, is associated with transformation and metastasis, which suggests that RSK is an attractive drug target. The natural product, SL0101 (kaempferol 3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside), has been shown to be a RSK selective inhibitor. However, the Ki for SL0101 is 1 μM with a half-life of less than 30 min in vivo. To identify analogues with improved efficacy we designed a set of analogues based on the crystallographic model of SL0101 in complex with the RSK2 N-terminal kinase domain. We identified an analogue with a 5″-n-propyl group on the rhamnose that has > 40-fold improved affinity for RSK relative to SL0101 in an in vitro kinase assay. This analogue preferentially inhibited the proliferation of the human breast cancer line, MCF-7, versus the normal untransformed breast line, MCF-10A, which is consistent with results using SL0101. However, the efficacy of the 5″-n-propyl analogue to inhibit MCF-7 proliferation was only two-fold better than for SL0101, which we hypothesize is due to limited membrane permeability. The improved affinity of the 5″-n-propyl analogue for RSK will aid in the design of future compounds for in vivo use.

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu(356) to Thr(370)) of the JD in the presence of calcium (Ca(2+)). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.

A Rising Cancer Prevention Target of RSK2 in Human Skin Cancer

RSK2 is a p90 ribosomal S6 kinase family (p90RSK) member regulating cell proliferation and transformation induced by tumor promoters such as epithelial growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate. This family of p90RSK has classified as a serine/threonine kinase that respond to many growth factors, peptide hormones, neurotransmitters, and environmental stresses such as ultraviolet (UV) light. Our recent study demonstrates that RSK2 plays a key role in human skin cancer development. Activation of RSK2 by EGF and UV through extracellular-activated protein kinases signaling pathway induces cell cycle progression, cell proliferation, and anchorage-independent cell transformation. Moreover, knockdown of RSK2 by si-RNA or sh-RNA abrogates cell proliferation and cell transformation of non-malignant human skin keratinocyte, and colony growth of malignant melanoma (MM) cells in soft agar. Importantly, activated and total RSK2 protein levels are highly detected in human skin cancer tissues including squamous cell carcinoma, basal-cell carcinoma, and MM. Kaempferol and eriodictyol are natural substances to inhibit kinase activity of the RSK2 N-terminal kinase domain, which is a critical kinase domain to transduce their activation signals to the substrates by phosphorylation. In this review, we discuss the role of RSK2 in skin cancer, particularly in activation of signaling pathways and potent natural substances to target RSK2 as chemopreventive and therapeutic agents.

RSK2Ser227 at N-Terminal Kinase Domain Is a Potential Therapeutic Target for Multiple Myeloma

Multiple myeloma is an entity of cytogenetically and genetically heterogenous plasma cell neoplasms. Despite recent improvement in the treatment outcome of multiple myeloma by novel molecular-targeted chemotherapeutics, multiple myeloma remains incurable. The identification of a therapeutic target molecule in which various signaling for cell-survival converge is a core component for the development of new therapeutic strategies against multiple myeloma. RSK2 is an essential mediator of the ERK1/2 signaling pathway for cell survival and proliferation. In this study, we discovered that RSK2(Ser227), which is located at the N-terminal kinase domain and is one site responsible for substrate phosphorylation, is activated through phosphorylation regardless of the type of cytogenetic abnormalities or upstream molecular signaling in all 12 multiple myeloma-derived cell lines examined and 6 of 9 patient-derived CD138-positive primary myeloma cells. The chemical inhibition of RSK2(Ser227) by BI-D1870 or gene knockdown of RSK2 inhibits myeloma cell proliferation through apoptosis induction, and this anti-myeloma effect was accompanied by downregulation of c-MYC, cyclin D, p21(WAF1/CIP1), and MCL1. RSK2(Ser227) inhibition resulting from BI-D1870 treatment restored lenalidomide-induced direct cytotoxicity of myeloma cells from interleukin-6-mediated cell protection, showed no cross-resistance to bortezomib, and exerted additive/synergistic antiproliferative effects in conjunction with the mTOR, histone deacetylase, and BH3-mimicking BCL2/BCLX(L) inhibitors. These results suggest that RSK2(Ser227) is a potential therapeutic target not only for newly diagnosed but also for patients with later phase multiple myeloma who are resistant or refractory to currently available anti-myeloma therapies.

Abstract B55: Involvement of RSK2 in human skin cancer

Abstract Our previous study demonstrated that the total p90 ribosomal S6 kinase 2 (RSK2) protein level was significantly higher in human skin cancer tissues and cancer cells compared with normal skin tissues and premalignant cell lines. We also demonstrated that the N-terminal kinase domain of RSK2 plays an important role in transducing the RSK2 activation signal to its substrates. RSK2 requires activation of its C-terminal domain by its upstream kinases, ERKs, in order to phosphorylate and activate its substrates. For example, a recombinant RSK2 protein purified from E. Coli cannot phosphorylate the RSK2 substrate, NFAT3, but phosphorylation of RSK2 by ERKs at the linker region of RSK2 gives RSK2 the ability to phosphorylate its substrates through its N-terminal kinase domain. This suggests that ERKs/RSK2 signaling plays an important role in RSK2-mediated cell proliferation and cell transformation. Importantly, our study demonstrated that co-expression of RasG12V and RSK2 in NIH3T3 cells increased foci formation compared with RasG12V alone and si-RNA knockdown of RSK2 in RasG12V- or RasG12V/RSK2-co-expressing NIH3T3 cells completely blocked foci formation. Furthermore, we demonstrated that kaempferol, a natural compound abundantly found in dietary foods, inhibited RSK2 N-terminal kinase activity and EGF-induced neoplastic cell transformation. These indicated that the Ras/ERKs/RSK2 signaling pathway plays a key role in cell transformation. However, the role of RSK2 activation and signaling in human skin cancer has not yet been fully elucidated. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2, but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. Moreover, RSK2−/− mouse embryonic fibroblasts (MEFs) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with UV. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. Citation Format: Cheol-Jung Lee, Mee Hyun Lee, Ke Yao, Hye Suk Lee, Ann M. Bode, Zigang Dong, Yong Yeon Cho. Involvement of RSK2 in human skin cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B55.