Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins βTrCP1 or βTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

The ribosomal S6 kinase 2 (RSK2), a member of the p90 RSK (RSK) family of proteins, is a widely expressed serine/ threonine kinase that is activated by extracellular signalregulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/ threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2 . These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA. [Cancer Res 2007;67(17):8104-12]

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by transduction of reprogramming factors, including Oct4, Sox2, Klf4, and c-Myc. A coordinated network of these factors was suggested to confer a pluripotency of iPSCs. Together with Oct4, Sox2 plays a major role as a master regulator in ESCs. However, the underlying mechanisms by which Sox2 contributes to selfrenewal or reprogramming processes remain to be determined. Here, we provide new evidence for a phosphorylation-based regulation of Sox2 activity. Akt directly interacts with Sox2 and promotes its stabilization through phosphorylation at Thr118, which enhances the transcriptional activity of Sox2 in ESCs. Moreover, phosphorylation of Sox2 cooperates in the reprogramming of mouse embryonic fibroblasts by enabling more efficient induction of iPSCs. Overall, our studies provide new insights into the regulatory mechanism of Sox2 in ESCs and also provide a direct link between phosphorylation events and somatic cell reprogramming.

Our previous findings indicated that RSK2 plays a critical role in proliferation and cell transformation induced by tumor promoters, such as epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate, and that kaempferol, a natural compound found in edible plants, selectively inhibits RSK2 activity. However, the molecular mechanism for RSK2 activation is unclear. Herein, we provide evidence showing that NH 2 -terminal kinase domain (NTD) activation of RSK2 is required for the activation of the extracellular signalregulated kinase-mediated COOH-terminal kinase domain (CTD). We also found that the NTD plays a key role in substrate phosphorylation and that kaempferol binds with the NTD but not the CTD in both the active and inactive forms. Homology modeling of the RSK2 NH 2 -terminal domain and small-molecule docking, validated by mutagenesis experiments, clearly showed that Val 82 and Lys 100 are critical amino acids for kaempferol binding and RSK2 activity. Furthermore, immunohistofluorescence and Western blot results indicated that the RSK2 protein level is markedly higher in cancer cell lines as well as cancer tissues compared with nonmalignant cell lines or normal tissues. In addition, kaempferol inhibited proliferation of malignant human cancer cell lines, including A431, SK-MEL-5 and SK-MEL-28, and HCT-116. These results indicate that targeting RSK2 with natural compounds, such as kaempferol, might be a good strategy for chemopreventive or chemotherapeutic application. [Cancer Res 2009;69(10):4398-406]

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

RSK2, an ERK downstream kinase, is a novel mediator of skeletal muscle cell differentiation through its regulation of NFAT3 activity. We found that the N-terminal (amino acids (aa) 1-68) and C-terminal (aa 416 -674) kinase domains of RSK2 directly interacted with nuclear localization signal 1, the Ser/Pro repeat, and the polyproline domains (aa 261-365) of NFAT3. Upon A23187 stimulation, RSK2 induced nuclear localization of NFAT3. RSK2 phosphorylated NFAT3 in vitro (K m ‫؍‬ 3.559 M), and activation of NFAT3 by RSK2 enhanced the promoter activity of NFAT3 downstream target genes in vivo. Furthermore, nuclear accumulation of NFAT3 was attenuated markedly in RSK2 ؊/؊ cells compared with wild-type RSK2 ؉/؉ cells. Notably, RSK2 and NFAT3 induced a significant differentiation of C2C12 myoblasts to multinucleated myotubes. Multinucleated myotube differentiation was inhibited by small interfering RNA against RSK2, ERK1/2, or NFAT3. These results demonstrate that RSK2 is an important kinase for NFAT3 in mediating myotube differentiation.Myoblast cell differentiation to muscle fibers depends on myogenic transcription factors, particularly myogenin (1). Many studies addressing the involvement of the mitogen-activated protein kinase (MAPK) 2 pathways are controversial because results showed both positive and negative effects of this signaling pathway on myogenesis. The MAPK cascades are implicated in the regulation of cell proliferation, survival, growth, and motility (2, 3) as well as tumorigenesis (4). Extracellular signal-regulated kinase (ERK)-1 and ERK2 (5, 6) mediate the 90-kDa ribosomal S6 kinases (RSKs), which are a family of broadly expressed serine/threonine kinases that respond to many growth factors, peptide hormones, and neurotransmitters (7,8). When activated, RSK2 translocates to the nucleus, where it can phosphorylate various nuclear proteins, including c-Fos, Elk-1, histones, and cAMP-responsive element-binding protein (CREB) (9 -13); activating transcription factor-4 (14); and p53 (15). Moreover, because RSKs have broad substrate specificity, RSK2 may be able to bind and/or phosphorylate a number of diverse substrates that regulate cell proliferation or differentiation or the cell cycle, depending on the specific situation.The nuclear factor of activated T cell (NFAT) family of transcription factors has been characterized primarily in immune cells (16). However, accumulating evidence has demonstrated that NFAT transcription factors are present in a wide range of cell types and tissues (17-22). NFAT3 and NFAT4 have an ϳ48% amino acid homology in the whole protein and exhibit an especially high degree of similarity (i.e. ϳ80%) in the Rel homology domain, which contains the DNA-binding motif. Moreover, the N-terminal domains (amino acids (aa) 1-360) of NFAT3 and NFAT4 have only a 30% amino acid similarity. Therefore, we proposed that the N-terminal domains of NFAT3 and NFAT4 might bind to different proteins, which might also phosphorylate key residues. c-Jun N-terminal kinase (JNK)-2 phosphorylates NFAT4, but...

The X-ray structure at 2.0-Å resolution of the p90 ribosomal S6 kinase 2 C-terminal kinase domain revealed a C-terminal autoinhibitory αL-helix that was embedded in the kinase scaffold and determines the inactive kinase conformation. We suggest a mechanism of activation through displacement of the αL-helix and rearrangement of the conserved residue Glu500, as well as the reorganization of the T-loop into the active conformation.The 90-kDa ribosomal S6 kinase 2 (RSK2) is broadly expressed in response to growth factors, peptide hormones, neurotransmitters, chemokines and other stimuli1 -3. RSK2 is a serine/ threonine kinase containing two distinct catalytically functional kinase domains connected by a linker region4 , 5. The C-terminal domain (CTD) phosphorylates the linker region 6 and regulates the N-terminal domain, which phosphorylates various substrates3 , 4 , 7 ,8 . No defined structure of RSK2 or of either kinase domain has been reported. However, sequence alignment with Ca 2+ /calmodulin-dependent kinase suggested the existence of an autoinhibitory helix outside the CTD RSK2 protein kinase domain, and its autoinhibitory role has been demonstrated in vivo 9 . Efficient activation of RSK2 requires interaction with extracellular signal-regulated protein kinases (ERKs) at a docking site in the RSK2 C terminus (residues 726-735) 10, 11 and subsequent phosphorylation of Thr577 in the CTD T-activation loop12.The CTD RSK2 adopted a classical bilobal kinase fold with an accessible catalytic cleft ( Fig. 1 and Supplementary Fig. 1 online). The C-terminal segment (residues 696-710) formed another αL-helix, located underneath the catalytic cleft and embedded in the kinase scaffold. It occupied a 'cradle' shaped by the αF and αG two-helix junction. The area of the αL-helix surface buried in the 'cradle' was ~800 Å 2 (~50% of the total area) and was composed mostly NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (~80%) of nonpolar atoms. The position of the αL-helix in the 'cradle' was stabilized by one hydrogen bond (2.7 Å) between Tyr707 and Ser603 (αF-helix).Superimposition of the structures of the RSK2 CTD and active cyclic AMP-dependent protein kinase (PKA) bound to an ATP analog and an inhibitory peptide ( Fig. 2a and Supplementary Fig. 2a online) showed that the CTD had a normal arrangement of the N and C lobes with a slightly more open cleft conformation than found in PKA. Comparing the CTD and PKA invariant residues showed that Asp539 (RD-motif), Asn544 (catalytic loop) and Asp561 (DFGmotif), as well as the ionic pair between Glu463 (αC-helix) and Lys451 (β3-strand), essentially adopted the same conformation in both the CTD and PKA and fit the requirements for optimal phospho-transfer 13 . Despite extensive efforts to cocrystallize the protein with an unhydrolyzable ATP analog, we could not obtain ATP-bound crystals, suggesting that the CTD adopts an inactive conformation.A notable difference between CTD RSK and active PKA entailed the position of the αD-helix, which was shi...

Blockade of the transient receptor potential channel vanilloid subfamily 1 (TRPV1) is suggested as a therapeutic approach to pain relief. However, TRPV1 is a widely expressed protein whose function might be critical in various nonneuronal physiologic conditions. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is overexpressed in many human epithelial cancers and is a potential target for anticancer drugs. Here, we show that TRPV1 interacts with EGFR, leading to EGFR degradation. Notably, the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis. The TRPV1 is the first membrane receptor shown to have a tumor-suppressing effect associated with the down-regulation of another membrane receptor. The data suggest that, although a great deal of interest has focused on TRPV1 as a target for pain relief, the chronic blockade of this pain receptor might increase the risk for cancer development. [Cancer Res 2009;69(3):905-13]