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J. Biol. Chem., Vol. 282, Issue 48, 34735-34747, November 30, 2007

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Evidence for the Direct Involvement of the Proteasome in the Proteolytic Processing of the Aspergillus nidulans Zinc Finger Transcription Factor PacC^*

América Hervás-Aguilar, José M. Rodríguez, Joan Tilburn, Herbert N. Arst, Jr., and Miguel A. Peñalva¹

From the Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid 28040, Spain and the Department of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom

The 72-kDa zinc finger transcription factor PacC, distantly related to Ci/Gli developmental regulators, undergoes two-step proteolytic processing in response to alkaline ambient pH. "Signaling protease" cleavage of PacC⁷² removes a processing-inhibitory C-terminal domain, making its truncated PacC⁵³ product accessible to a second "processing" protease, yielding PacC²⁷. Features of the processing proteolysis suggested the proteasome as a candidate protease. We constructed, using gene replacements, two missense active site mutations in preB, the Aspergillus nidulans orthologue of Saccharomyces cerevisiae PRE2 encoding the proteasome β5 subunit. preB1^K101A is lethal. Viable preB2^K101R impairs growth and, like its equivalent pre2^K108R in yeast, impairs chymotryptic activity. pre2^K108R and preB2^K101R active site mutations consistently shift position of the scissile bonds when PacC is processed in S. cerevisiae and A. nidulans, respectively, indicating that PacC must be a direct substrate of the proteasome. preB2^K101R leads to a 2–3-fold elevation in NimE mitotic cyclin levels but appears to result in PacC instability, suggesting an altered balance between processing and degradation. preB2^K101R compensates the marked impairment in PacC²⁷ formation resulting from deletion of the processing efficiency determinant in PacC, further indicating direct proteasomal involvement in the formation of PacC²⁷. Deletion of a Gly-Pro-Ala-rich region within this processing efficiency determinant markedly destabilizes PacC. Arg substitutions of Lys residues within this efficiency determinant and nearby show that they cooperate to promote PacC processing. A quadruple Lys-to-Arg substitution (4KR) impairs formation of PacC²⁷ and leads to persistence of PacC⁵³. Wild-type PacC⁵³ becomes multiply phosphorylated upon alkaline pH exposure. Processing-impaired 4KR PacC⁵³ becomes excessively phosphorylated.

Received for publication, August 13, 2007 , and in revised form, September 27, 2007.

^* This work was supported by Dirección General de Investigación Científica y Técnica (Ministerio de Educación y Ciencia, Spain) Grants BIO2003-0077 and BIO2006-556 (to M. A. P.) and Wellcome Trust Grant 067878 (to H. N. A. and J. T.). 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 Figs. S1–S6.

¹ To whom correspondence should be addressed: Centro de Investigaciones Biológicas del CSIC, Ramiro de Maeztu 9, Madrid 29040, Spain. Tel.: 34918373112 (ext. 4358); Fax: 34915360432; E-mail: penalva{at}cib.csic.es.

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