Abstract
STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cytokine-inducible SH2-containing protein (cish). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation.
Highlights
STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown
Because STAT5 has a prominent role in adipocyte development and function, we sought to identify novel STAT5-interacting proteins in fat cells to further understand the role of this signaling molecule and transcription factor
Both of these proteins, pyruvate dehydrogenase complex (PDC)-E1 and PDC-E2, interacted with STAT5A in a growth hormone (GH)-regulated manner, suggesting that STAT5 only interacts with the PDC complex when it is activated by tyrosine phosphorylation
Summary
To gain insight into the potential mechanisms involved in the ability of STAT5 to modulate fat cell function, we employed a non-biased MS-based approach to identify novel STAT5-interacting proteins. We performed this experiment with whole-cell extracts from human adipocytes (Fig. 1B) and again observed a GH-dependent interaction between STAT5A and PDC-E2. Whole-cell extracts were incubated with a STAT5A antibody, and associated proteins pulled down by immunoprecipitation were examined by Western blotting (left-hand side of Fig. 2). Western blotting was used to examine the protein content of the immunoprecipitates (IP: STAT5A; left) and homogenate inputs (Direct WB; 150 g of protein/lane; right) This experiment was performed one time, and two mice per condition were examined. We observed that this coactivator bound the cish promoter at levels very similar to what we observed for PDC-E2
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