TY - JOUR
T1 - Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins
AU - Sun, Liang
AU - Prince, Thomas
AU - Manjarrez, Jacob R.
AU - Scroggins, Bradley T.
AU - Matts, Robert L.
N1 - Funding Information:
The authors would like to thank Dr. Carolyn Machamer (Johns Hopkins University School of Medicine) for generously providing the anti-Aha1/p38 antibody. Geldanamycin was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH. The anti-p23, HOP, HIP, and Hsp70 antibodies were kindly provided by Dr. David Toft (Mayo Medical School, Rochester, MN) and Dr. David Smith (Mayo Clinic, Scottsdale, AZ). We would also like to thank Dr. Steven Hartson (Director, OSU DNA/Protein Core Facility) for his help with the LC–MS/MS analysis. This work was supported by the Oklahoma Agricultural Experiment Station ( Project 1975 ) and by grant number HR03-076 from the Oklahoma Center for the Advancement of Science and Technology (to R.L.M.).
PY - 2012/6
Y1 - 2012/6
N2 - The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.
AB - The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.
KW - Activator of Hsp90 ATPase
KW - Aha1-interacting protein
KW - Heat shock protein 90
KW - Hsp90 inhibitor
KW - Non-receptor tyrosine kinase v-Src
KW - Progesterone receptor
UR - http://www.scopus.com/inward/record.url?scp=84860356248&partnerID=8YFLogxK
U2 - 10.1016/j.bbamcr.2012.03.014
DO - 10.1016/j.bbamcr.2012.03.014
M3 - Article
C2 - 22504172
AN - SCOPUS:84860356248
SN - 0167-4889
VL - 1823
SP - 1092
EP - 1101
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
IS - 6
ER -