Reversible acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a major role in transcriptional regulation. HATs promote transcriptional activation by adding acetyl groups to the histone tails, while HDACs act in the reverse process leading to transcriptional repression. In addition to histones, HATs and HDACs also modify numerous proteins, and therefore affect many biological processes. The HDAC family consists of 18 members, which are grouped into four classes, namely I, II, III and IV, with class II further subdivided into subclass IIa and IIb. HDAC6 is a predominantly localized cytoplasmic deacetylase, which belongs to class IIb. HDAC6 is unique among the HDAC family members in that it expresses two complete functional deacetylase domains, a cytoplasmic anchoring motif, and an ubiquitin binding domain. Like many other HDACs, HDAC6 has a growing list of substrates. HDAC6 is known for its deacetylase activity towards cytoplasmic proteins α-tubulin and cortactin, a function that is known to promote HDAC6- mediated cell migration. HDAC6 is also regulated by post translational modifications such as, phosphorylation, acetylation and ubiquitination. Using mass spectrometric analysis, we have identified additional phosphorylation sites, Threonine-1031 and Serine-1035, within the C-terminal of HDAC6. Both sites are located within an extracellular signal regulated kinase (ERK) recognition motif, while Serine-1035 is a major site targeted by ERK. Previous studies have identified ERK as a pivotal player in promoting tumorgenesis. HDAC6 was identified as a major player in promoting cell migration, and was also shown to be required for efficient oncogenic tumorigenesis. However, whether ERK can regulate HDAC6 to promote cell migration have not been investigated. Based on published data and our preliminary results, we hypothesized that 1) Serine-1035 of HDAC6 is a major phosphorylation site of HDAC6 that plays a critical role in promoting HDAC6-mediated cell migration by affecting deacetylation of its substrates; and 2) HDAC6 promotes cell migration in vivo. Using in vitro and in vivo studies, we confirmed that Serine-1035 of HDAC6 is phosphorylated by ERK1. In addition, we found that activation of the ERK-mitogen activated protein kinase (MAPK) signaling axis promotes HDAC6 phosphorylation. By using unphosphorylation mimicking mutants of HDAC6 Serine-1035 site, we observed that unphosphorylation of HDAC6 at this site does not affect HDAC6 enzymatic activity towards histones or cortactin. However, unphosphorylation of HDAC6 at Serine-1035 hinders HDAC6’s ability to deacetylate α-tubulin and β-catenin. Importantly, hypo-phosphorylation of HDAC6 at this site significantly attenuates its ability to promote cell migration using the cell lines model. We showed for the first time that HDAC6 also promotes metastasis in vivo using a mouse model. In conclusion, this study identified HDAC6 Serine-1035 as a novel phosphorylation site which can be phosphorylated by ERK1, and this phosphorylation status of HDAC6 can promote cell migration. Since the ERK-MAPK pathway is a major target of numerous signaling events, inhibitors of both ERK and HDAC6 may prove beneficial for future research directions.
|Advisor:||Zhang, Xiaohong Mary|
|Commitee:||Kruk, Patricia, Nicosia, Santo, Seto, Edward, Wu, Jie|
|School:||University of South Florida|
|School Location:||United States -- Florida|
|Source:||DAI-B 74/12(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry|
|Keywords:||Deacetylation, Extracellular signal regulated kinase 1, Hdac6, Histone, Phosphorylation|
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