Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

Abstract

Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (proliferating cell nuclear antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNAF/F) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNAF/F MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNAF/F mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.

Maternal diethylhexyl phthalate exposure affects adiposity and insulin tolerance in offspring in a PCNA-dependent manner

“…Antibodies for analysis included: anti-PCNA (Santa Cruz), anti-PPARγ (Cell Signaling Technologies), anti-actin C4 antibody (Abcam), and anti-Phospho Y114 PCNA generated via rabbits immunized with a KLH-conjugated peptide (synthetic sequence CNQEKVSD-pY-EMKLMD; Yenzym) and subsequent isolation of serum and extraction via a phosphopeptide affinity matrix and clean-up with an affinity matrix conjugated to unphosphorylated peptides (Lo, Ho et al 2013). Diethylhexyl phthalate (DEHP), benzylbutyl phthalate (BBP), diisononyl phthalate (DNP) and Oil Red O were purchased from Sigma.…”

“…Mouse embryonic fibroblasts (MEFs) used for in vitro studies were previously described (Lo, Ho et al 2013). After plating MEFs and allowing for attachment, media with or without 0.5 μM DEHP was utilized for the totality of the adipogenesis protocol as previously described (Tang, Otto et al 2003).…”

“…Wild type (WT/WT) mice and mice with a point mutation of PCNA at residue 114 which changes tyrosine to phenylalanine (PCNA Y114F/Y114F )(Lo, Ho et al 2013) in a FVB background were maintained per approved University of Cincinnati Institutional Animal Care and Use Committee (IACUC) protocols. Mice genotypes were confirmed as previously described (Lo, Ho et al 2013).…”

“…Previously, our laboratory has shown that phosphorylation (p) of proliferating cell nuclear antigen (PCNA) at tyrosine 114 (Y114) plays a significant role in adipocyte maturation and subsequent clonal expansion in mice (Lo, Ho et al 2013). When placed on a high-fat diet (HFD), mice defective for Y114 phosphorylation on PCNA exhibit a less obese phenotype compared to the wild type mice over a prolonged time course, establishing a role of Y114 phosphorylation (p-Y114) of PCNA to HFD-induced obesity.…”

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis

“…The association with ERK8 kinase also influenced PCNA stability by regulating the interaction with MDM2, although no evidence that ERK8 could phosphorylate PCNA, was provided (29). Finally, Tyr114 phosphorylation has been recently reported to control adipocytes generation (30). …”