Phosphorylation regulates human OCT4

Abstract

The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.proteomics | posttranslational regulation O CT4 is a homeobox transcription factor that was first identified for its essential role in early mammalian development (1-3). It is expressed in totipotent and pluripotent cells and down-regulated on differentiation (4-6). OCT4 is required to maintain pluripotency both in vivo and in cell culture (2,7,8), and it is indispensable for transcription factor-mediated reprogramming (9-12). Together with NANOG and SOX2, OCT4 carries out these functions by activating transcription of genes that support pluripotency and repressing genes involved in development (13)(14)(15)(16)(17).A variety of proteins, including NANOG, SOX2, and OCT4 itself, form an intricate regulatory loop that balances OCT4 expression (15,(18)(19)(20). Genomic and epigenetic studies have identified additional mechanisms that directly or indirectly influence OCT4 expression, providing a detailed view of its transcriptional regulation (21-26). OCT4 function is closely tied to its regulation, because decreasing OCT4 mRNA levels cause differentiation into trophoblast, and increasing OCT4 expression by as little as 1.5-fold causes differentiation to primitive endoderm (7). The relative stoichiometry of OCT4 and SOX2 is also important for establishing pluripotency, because the efficiency of reprogramming is dependent on the proportion of OCT4 and SOX2 transcripts (9). Thus, precise regulation of OCT4 is essential for pluripotency.Although OCT4 transcriptional regulation has been extensively studied, far less is known about its posttranslational regulation. Previous studies have speculated that phosphorylation controls OCT4 activity (27,28). For example, differences in electrophoretic mobility suggested that the homeobox domain of OCT4 is differentially phosphorylated when expressed in 293 cells compared with HeLa cells (27). Intriguingly, these different states correlated with OCT4's ability to acti...

Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4

“…39,40 By combining in vitro phosphorylation and phosphoproteomic analyses, we were able to identify several novel putative AKT phosphorylation sites in the OSKM factors with the exception of c-MYC. For OCT4 we confirmed not only the previously reported AKT phosphorylation site T235, 29,33 but also found new phosphorylation sites at S136, T159, T225 and S236. Likewise, for KLF4 we identified the reported AKT target site at S429 31 as well as novel phosphorylation sites at S19, T33, S234 and S326.…”

“…In contrast to our study, previous in vitro phosphorylation studies used recombinant transcription factors expressed in bacteria. 29,33 Bacterial expression systems exhibit several limitations, because the recombinant proteins are mostly localized in inclusion bodies, requiring protein denaturation and an often inefficient refolding. 34 In line with this notion, we found that several commercial preparations of bacterially expressed OSKM factors lacked robust DNA-binding activity.…”

“…Previous in vitro phosphorylation studies have been solely performed with recombinant OSKM proteins expressed in bacteria. 29,33 This approach exhibits several limitations, because the transcription factors are mostly expressed in inclusion bodies and need to be denatured and refolded in vitro. However, refolding of proteins from inclusion bodies is challenging and usually results in poor yields of natively folded, bioactive proteins.…”

Effects of siRNA-mediated silencing of Sal-like 4 expression on proliferation and apoptosis of prostate cancer C4-2 cells

“…Since phosphorylated ERK may initiate rapid proliferation of tumor cells by activating SALL4 after entering cells, high SALL4 expression may be regulated by prostate-specific membrane antigen in prostate cancer (Brumbaugh et al, 2012). Currently, the regulatory effects of SALL4 on the biological functions of prostate cancer cells have never been reported.…”

Stem cell powwow in Squaw Valley

“…However, Thomson's main focus was on phosphorylation of the core pluripotency factors; using improved mass spectrometry, Thomson's group have identified 14 phosphorylation sites in Oct4 and six in Sox2 (Brumbaugh et al, 2012). Phosphorylation at two of the identified sites in Oct4, both of which lie within the homeodomain, decreases Oct4 DNA binding and transcriptional activity.…”