Protein Stability

Preventing Interaction with the Proteasome

Science Signaling  04 Mar 2008:
Vol. 1, Issue 9, pp. ec82
DOI: 10.1126/stke.19ec82

Steroid receptor coactivator 3 (SRC-3) interacts with estrogen receptors to promote estrogen-mediated transcription and genetic amplification, and overexpression of SRC-3 has been associated with various cancers, including breast cancer. Phosphorylation by p38 mitogen-activated protein kinase or glycogen synthase kinase 3β has been associated with enhanced degradation of SRC-3. Yi et al. sought to determine whether other kinases regulated SRC-3 activity or abundance and, through cotransfection experiments, identified atypical protein kinase C, both aPKCζ and PKCι, as kinases that when coexpressed with tagged SRC-3 and the estrogen receptor ERα increased the abundance of SRC-3. The stabilized SRC-3 was functional and stimulated estrogen-dependent gene expression both in transfected cells and in an in vitro transcription assay. Knockdown experiments with siRNAs confirmed that endogenous SRC-3 abundance was decreased in cells in which abundance of both aPKCs was decreased. The ability of aPKCζ to increase the abundance of SRC-3 depended on ERα and estrogen and did not occur when the cells were cotransfected with the ERβ estrogen receptor. Coimmunoprecipitation experiments showed that both ERα and SRC-3 interacted with aPKCζ. Although the ability of aPKCζ to stabilize SRC-3 required ERα and estrogen and SRC-3 mutants that did not bind ERα were not stabilized by aPKCζ, the interaction between SRC-3 and aPKCζ did not require the presence of ERα. Instead, in vitro kinase assays suggested that ERα promoted the phosphorylation of SRC-3 by aPKCζ. Mutation analysis indicated that the aPKC-mediated stabilization of SRC-3 involved an acidic region in the C-terminal region of SRC-3. SRC-3 interacted with the C8 subunit of the 20S proteasome in glutathione S-transferase pull-down experiments, and SRC-3 isolated from cells cotransfected with ERα and aPKCζ exhibited reduced interaction with this proteasomal subunit. Proteasomal degradation can be mediated by ubiquitin-dependent and -independent mechanisms, the latter involving a REGγ complex. In vitro assays for ubiquitin-dependent or REGγ-dependent proteasomal degradation showed that SRC-3 from cells cotransfected with ERα and aPKCζ was resistant to degradation. The authors suggest that phosphorylation of SRC-3 by aPKCs leads to a conformational change that exposes the acidic region and prevents the interaction between SRC-3 and the C8 proteasomal subunit.

P. Yi, Q. Feng, L. Amazit, D. M. Lonard, S. Y. Tsai, M.-J. Tsai, B. W. O'Malley, Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1. Mol. Cell 29, 465-476 (2008). [PubMed]