A unique and highly conserved structural feature of 90-kDa ribosomal S6 kinase (p90 or RSK) is the presence of two non-identical kinase domains. To explore the mechanism of RSK activation, a cloned human RSK cDNA (RSK3) was used to generate and characterize several site-directed RSK mutants: K91A (N-Lys, NH-terminal ATP-binding mutant), K444A (C-Lys, COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant), T570A (C-Thr, mutant of the putative MAPK phosphorylation site in subdomain VIII of the C-domain), S218A (N-Ser, mutant of the corresponding NH-terminal residue). Epitope-tagged RSKs were expressed in transfected COS cells followed by immunoprecipitation with or without prior in vivo epidermal growth factor stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was ablated (and partially impaired with N-Ser). In contrast, both C-Lys and C-Thr retained high levels of kinase activity and were capable of responding to stimulation. C-Lys also retained partial kinase activity toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1 G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)) whereas NLys did not. The isolated NH- and COOH-terminal domains were also expressed; the C-domain was inactive, whereas the N-domain retained partial activity. Relative to wild-type, both N-Lys and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein tyrosine phosphatase. We conclude that 1) the NH-terminal RSK kinase domain mediates substrate phosphorylation; 2) both domains contribute to autophosphorylation; 3) the putative MAPK phosphorylation site is not required for growth factor-stimulated autophosphorylation or kinase activation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Thakur, Y. Sun, A. Bollig, J. Wu, H. Biliran, S. Banerjee, F. H. Sarkar, and D. J. Liao Anti-invasive and Antimetastatic Activities of Ribosomal Protein S6 Kinase 4 in Breast Cancer Cells Clin. Cancer Res., July 15, 2008; 14(14): 4427 - 4436. [Abstract] [Full Text] [PDF]

				H. Deng, X. Bao, W. Cai, M. J. Blacketer, A. S. Belmont, J. Girton, J. Johansen, and K. M. Johansen Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila Development, February 15, 2008; 135(4): 699 - 705. [Abstract] [Full Text] [PDF]

				J.-Q. Dai, X.-J. Zhu, F.-Q. Liu, J.-H. Xiang, H. Nagasawa, and W.-J. Yang Involvement of p90 Ribosomal S6 Kinase in Termination of Cell Cycle Arrest during Development of Artemia-encysted Embryos J. Biol. Chem., January 18, 2008; 283(3): 1705 - 1712. [Abstract] [Full Text] [PDF]

				J.-X. Lin, R. Spolski, and W. J. Leonard Critical role for Rsk2 in T-lymphocyte activation Blood, January 15, 2008; 111(2): 525 - 533. [Abstract] [Full Text] [PDF]

				M. Ikuta, M. Kornienko, N. Byrne, J. C. Reid, S. Mizuarai, H. Kotani, and S. K. Munshi Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors Protein Sci., December 1, 2007; 16(12): 2626 - 2635. [Abstract] [Full Text] [PDF]

				D. Chaturvedi, H. M. Poppleton, T. Stringfield, A. Barbier, and T. B. Patel Subcellular Localization and Biological Actions of Activated RSK1 Are Determined by Its Interactions with Subunits of Cyclic AMP-Dependent Protein Kinase Mol. Cell. Biol., June 15, 2006; 26(12): 4586 - 4600. [Abstract] [Full Text] [PDF]

				N. Maekawa, J.-i. Abe, T. Shishido, S. Itoh, B. Ding, V. K. Sharma, S.-S. Sheu, B. C. Blaxall, and B. C. Berk Inhibiting p90 Ribosomal S6 Kinase Prevents Na+-H+ Exchanger-Mediated Cardiac Ischemia-Reperfusion Injury Circulation, May 30, 2006; 113(21): 2516 - 2523. [Abstract] [Full Text] [PDF]

				B. A. Dummler, C. Hauge, J. Silber, H. G. Yntema, L. S. Kruse, B. Kofoed, B. A. Hemmings, D. R. Alessi, and M. Frodin Functional Characterization of Human RSK4, a New 90-kDa Ribosomal S6 Kinase, Reveals Constitutive Activation in Most Cell Types J. Biol. Chem., April 8, 2005; 280(14): 13304 - 13314. [Abstract] [Full Text] [PDF]

				J. A. Smith, C. E. Poteet-Smith, Y. Xu, T. M. Errington, S. M. Hecht, and D. A. Lannigan Identification of the First Specific Inhibitor of p90 Ribosomal S6 Kinase (RSK) Reveals an Unexpected Role for RSK in Cancer Cell Proliferation Cancer Res., February 1, 2005; 65(3): 1027 - 1034. [Abstract] [Full Text] [PDF]

				G. Putz, F. Bertolucci, T. Raabe, T. Zars, and M. Heisenberg The S6KII (rsk) Gene of Drosophila melanogaster Differentially Affects an Operant and a Classical Learning Task J. Neurosci., November 3, 2004; 24(44): 9745 - 9751. [Abstract] [Full Text] [PDF]

				P. P. Roux and J. Blenis ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions Microbiol. Mol. Biol. Rev., June 1, 2004; 68(2): 320 - 344. [Abstract] [Full Text] [PDF]

				A. P. Myers, L. B. Corson, J. Rossant, and J. C. Baker Characterization of Mouse Rsk4 as an Inhibitor of Fibroblast Growth Factor-RAS-Extracellular Signal-Regulated Kinase Signaling Mol. Cell. Biol., May 15, 2004; 24(10): 4255 - 4266. [Abstract] [Full Text] [PDF]

				K. M. Eisenmann, M. W. VanBrocklin, N. A. Staffend, S. M. Kitchen, and H.-M. Koo Mitogen-Activated Protein Kinase Pathway-Dependent Tumor-Specific Survival Signaling in Melanoma Cells through Inactivation of the Proapoptotic Protein Bad Cancer Res., December 1, 2003; 63(23): 8330 - 8337. [Abstract] [Full Text] [PDF]

				H. Vaidyanathan and J. W. Ramos RSK2 Activity Is Regulated by Its Interaction with PEA-15 J. Biol. Chem., August 22, 2003; 278(34): 32367 - 32372. [Abstract] [Full Text] [PDF]

				P. P. Roux, S. A. Richards, and J. Blenis Phosphorylation of p90 Ribosomal S6 Kinase (RSK) Regulates Extracellular Signal-Regulated Kinase Docking and RSK Activity Mol. Cell. Biol., July 15, 2003; 23(14): 4796 - 4804. [Abstract] [Full Text] [PDF]

				K. El-Haschimi, S. D. Dufresne, M. F. Hirshman, J. S. Flier, L. J. Goodyear, and C. Bjorbaek Insulin Resistance and Lipodystrophy in Mice Lacking Ribosomal S6 Kinase 2 Diabetes, June 1, 2003; 52(6): 1340 - 1346. [Abstract] [Full Text] [PDF]

				H.-Y. Fan, C. Tong, L. Lian, S.-W. Li, W.-X. Gao, Y. Cheng, D.-Y. Chen, H. Schatten, and Q.-Y. Sun Characterization of Ribosomal S6 Protein Kinase p90rsk During Meiotic Maturation and Fertilization in Pig Oocytes: Mitogen-Activated Protein Kinase-Associated Activation and Localization Biol Reprod, March 1, 2003; 68(3): 968 - 977. [Abstract] [Full Text] [PDF]

				A. M. Hinsby, J. V. Olsen, K. L. Bennett, and M. Mann Signaling Initiated by Overexpression of the Fibroblast Growth Factor Receptor-1 Investigated by Mass Spectrometry Mol. Cell. Proteomics, January 1, 2003; 2(1): 29 - 36. [Abstract] [Full Text] [PDF]

				C. A. Chrestensen and T. W. Sturgill Characterization of the p90 Ribosomal S6 Kinase 2 Carboxyl-terminal Domain as a Protein Kinase J. Biol. Chem., July 26, 2002; 277(31): 27733 - 27741. [Abstract] [Full Text] [PDF]

				K. Merienne, S. Pannetier, A. Harel-Bellan, and P. Sassone-Corsi Mitogen-Regulated RSK2-CBP Interaction Controls Their Kinase and Acetylase Activities Mol. Cell. Biol., October 15, 2001; 21(20): 7089 - 7096. [Abstract] [Full Text] [PDF]

				S. D. Dufresne, C. Bjørbæk, K. El-Haschimi, Y. Zhao, W. G. Aschenbach, D. E. Moller, and L. J. Goodyear Altered Extracellular Signal-Regulated Kinase Signaling and Glycogen Metabolism in Skeletal Muscle from p90 Ribosomal S6 Kinase 2 Knockout Mice Mol. Cell. Biol., January 1, 2001; 21(1): 81 - 87. [Abstract] [Full Text]

				J. C. Bruning, J. A. Gillette, Y. Zhao, C. Bjorbaeck, J. Kotzka, B. Knebel, H. Avci, B. Hanstein, P. Lingohr, D. E. Moller, et al. Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene PNAS, March 14, 2000; 97(6): 2462 - 2467. [Abstract] [Full Text] [PDF]

				M. A. Stevenson, M.-J. Zhao, A. Asea, C. N. Coleman, and S. K. Calderwood Salicylic Acid and Aspirin Inhibit the Activity of RSK2 Kinase and Repress RSK2-Dependent Transcription of Cyclic AMP Response Element Binding Protein- and NF-{kappa}B-Responsive Genes J. Immunol., November 15, 1999; 163(10): 5608 - 5616. [Abstract] [Full Text] [PDF]

				S. D. Gross, M. S. Schwab, A. L. Lewellyn, and J. L. Maller Induction of Metaphase Arrest in Cleaving Xenopus Embryos by the Protein Kinase p90Rsk Science, November 12, 1999; 286(5443): 1365 - 1367. [Abstract] [Full Text]

				C. J. Jensen, M.-B. Buch, T. O. Krag, B. A. Hemmings, S. Gammeltoft, and M. Frodin 90-kDa Ribosomal S6 Kinase Is Phosphorylated and Activated by 3-Phosphoinositide-dependent Protein Kinase-1 J. Biol. Chem., September 17, 1999; 274(38): 27168 - 27176. [Abstract] [Full Text] [PDF]

				A.-C. Gavin, A. N. Ainle, E. Chierici, M. Jones, and A. R. Nebreda A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes Mol. Biol. Cell, September 1, 1999; 10(9): 2971 - 2986. [Abstract] [Full Text]

				A. N. Moor and L. Fliegel Protein Kinase-mediated Regulation of the Na+/H+ Exchanger in the Rat Myocardium by Mitogen-activated Protein Kinase-dependent Pathways J. Biol. Chem., August 13, 1999; 274(33): 22985 - 22992. [Abstract] [Full Text] [PDF]

				C. E. Poteet-Smith, J. A. Smith, D. A. Lannigan, T. A. Freed, and T. W. Sturgill Generation of Constitutively Active p90 Ribosomal S6 Kinase in Vivo. IMPLICATIONS FOR THE MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATED PROTEIN KINASE FAMILY J. Biol. Chem., August 6, 1999; 274(32): 22135 - 22138. [Abstract] [Full Text] [PDF]

				H.-L. Li, W. Davis, and E. Pure Suboptimal Cross-linking of Antigen Receptor Induces Syk-dependent Activation of p70S6 Kinase through Protein Kinase C and Phosphoinositol 3-Kinase J. Biol. Chem., April 2, 1999; 274(14): 9812 - 9820. [Abstract] [Full Text] [PDF]

				K. D. Swanson, L. K. Taylor, L. Haung, A. L. Burlingame, and G. E. Landreth Transcription Factor Phosphorylation by pp90rsk2. IDENTIFICATION OF Fos KINASE AND NGFI-B KINASE I AS pp90rsk2 J. Biol. Chem., February 5, 1999; 274(6): 3385 - 3395. [Abstract] [Full Text] [PDF]

				D. J. Spring and E. G. Krebs Deletion of 11 Amino Acids in p90rsk-mo-1 Abolishes Kinase Activity Mol. Cell. Biol., January 1, 1999; 19(1): 317 - 320. [Abstract] [Full Text] [PDF]

				D. De Cesare, S. Jacquot, A. Hanauer, and P. Sassone-Corsi Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene PNAS, October 13, 1998; 95(21): 12202 - 12207. [Abstract] [Full Text] [PDF]

				P. B. Joel, J. Smith, T. W. Sturgill, T. L. Fisher, J. Blenis, and D. A. Lannigan pp90rsk1 Regulates Estrogen Receptor-Mediated Transcription through Phosphorylation of Ser-167 Mol. Cell. Biol., April 1, 1998; 18(4): 1978 - 1984. [Abstract] [Full Text]

				K. N. Dalby, N. Morrice, F. B. Caudwell, J. Avruch, and P. Cohen Identification of Regulatory Phosphorylation Sites in Mitogen-activated Protein Kinase (MAPK)-activated Protein Kinase-1a/p90rsk That Are Inducible by MAPK J. Biol. Chem., January 16, 1998; 273(3): 1496 - 1505. [Abstract] [Full Text] [PDF]

				C. M. Moxham, A. Tabrizchi, R. J. Davis, and CraigC. Malbon jun N-terminal Kinase Mediates Activation of Skeletal Muscle Glycogen Synthase by Insulin in Vivo J. Biol. Chem., November 29, 1996; 271(48): 30765 - 30773. [Abstract] [Full Text] [PDF]

				Y. Zhao, C. Bjorbak, and D. E. Moller Regulation and Interaction of pp90rsk Isoforms with Mitogen-activated Protein Kinases J. Biol. Chem., November 22, 1996; 271(47): 29773 - 29779. [Abstract] [Full Text] [PDF]

				E. V. Wong, A. W. Schaefer, G. Landreth, and V. Lemmon Involvement of p90rsk in Neurite Outgrowth Mediated by the Cell Adhesion Molecule L1 J. Biol. Chem., July 26, 1996; 271(30): 18217 - 18223. [Abstract] [Full Text] [PDF]

				M. Tomas-Zuber, J.-L. Mary, and W. Lesslauer Control Sites of Ribosomal S6 Kinase B and Persistent Activation through Tumor Necrosis Factor J. Biol. Chem., July 28, 2000; 275(31): 23549 - 23558. [Abstract] [Full Text] [PDF]

				J. A. Smith, C. E. Poteet-Smith, D. A. Lannigan, T. A. Freed, A. J. Zoltoski, and T. W. Sturgill Creation of a Stress-activated p90 Ribosomal S6 Kinase. THE CARBOXYL-TERMINAL TAIL OF THE MAPK-ACTIVATED PROTEIN KINASES DICTATES THE SIGNAL TRANSDUCTION PATHWAY IN WHICH THEY FUNCTION J. Biol. Chem., October 6, 2000; 275(41): 31588 - 31593. [Abstract] [Full Text] [PDF]

				R. R. Bhatt and J. E. Ferrell Jr. Cloning and Characterization of Xenopus Rsk2, the Predominant p90 Rsk Isozyme in Oocytes and Eggs J. Biol. Chem., October 13, 2000; 275(42): 32983 - 32990. [Abstract] [Full Text] [PDF]