Tertiary Structural Rearrangements upon Oxidation of Methionine145 in Calmodulin Promotes Targeted Proteasomal Degradation
Colette A Sacksteder 1, Jennifer E Whittier 1, Yijia Xiong 1, Jinhui Li 1, Nadezhda Galeva 2, Michael E Jacoby 1, Samuel Purvine 1, Todd D Williams 2, Martin C Rechsteiner 3, Diana J. Bigelow 4 and Thomas C Squier 1*
1 Pacific Northwest National Laboratory
2 University of Kansas
3 University of Utah
4 Pacific NW National Laboratory
* To whom correspondence should be addressed. E-mail: thomas.squier{at}pnl.gov.
Submitted on March 29, 2006
Revised on May 1, 2006
Accepted on 19 May 2006
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Abstract |
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The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides [Met(O)], suggesting a preferential degradation around Met(O) within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines for oxidation. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM [Anbanandam A., Bieber Urbauer R.J., Bartlett R.K., Smallwood H.S., Squier T.C., and Urbauer J.L. (2005) Biochemistry 44, 9486-9496], permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant CD spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.
Key Words:
Calcium Regulation, Cell Signaling, Methionine Sensors, Oxidative Stress, Protein Degradation, Redox Metabolism
