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J. Biol. Chem., Vol. 283, Issue 25, 17450-17462, June 20, 2008

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The Functional Interaction of 14-3-3 Proteins with the ERK1/2 Scaffold KSR1 Occurs in an Isoform-specific Manner^*

Lucas R. Jagemann¹, Luís G. Pérez-Rivas², E. Josué Ruiz³, Juan A. Ranea⁴, Francisca Sánchez-Jiménez^¶, Ángel R. Nebreda, Emilio Alba^||, and José Lozano⁵

From the Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain, the ^¶Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 29071 Málaga, Spain, the Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain, and ^||Servicio de Oncología Médica, Hospital Clínico Universitario Virgen de la Victoria, 29071 Málaga, Spain

Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3 with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3. Specific binding to 14-3-3 protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3 resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3 being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3 heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation.

Received for publication, November 8, 2007 , and in revised form, March 24, 2008.

^* This work was supported in part by Ministerio de Educación y Ciencia (MEC) Grants BMC2003-01607 and BFU06-01209 (to J. L.) and BFU2004-03566 (to A. R. N.) and by funds from Plan Andaluz de Investigación (to F. S.-J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1–3.

¹ Supported by funds from MEC and Fundación Instituto Mediterráneo para el Avance de la Biotecnología y la Investigación Sanitaria (IMABIS).

² Supported by a FPU predoctoral fellowship from MEC.

³ Supported by a FPI predoctoral fellowship from MEC.

⁴ A Ramón y Cajal investigator.

⁵ To whom correspondence should be addressed: Dpto. de Biología Molecular y Bioquímica, Universidad de Málaga. Campus de Teatinos s/n 29071 Málaga, Spain. Tel.: 34-95-213-6661; Fax: 34-95-213-2000; E-mail: jlozano{at}uma.es.

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