Tyrosine phosphorylation controls PCNA function through protein stability.

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

Analysis of luteal tissue inhibitor of metalloproteinase-1, -2, and -3 during prostaglandin F(2alpha)-induced luteolysis.

Increased matrix metalloproteinase (MMP) expression and activities help to mediate tissue involution through increasing extracellular matrix remodeling and promoting dedifferentiation and, ultimately, apoptosis. Therefore, we hypothesized that prostaglandin (PG) F(2alpha) administration would decrease expression of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3 and effectively increase the MMP:TIMP ratio, leading to glandular involution. In experiment 1, we tested the effects of PGF(2alpha) administration (Day 10 postestrus; Day 0 = estrus) on luteal TIMP-1, -2, and -3 mRNA and protein expression. Corpora lutea were collected at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, and 48 h following PGF(2alpha) administration (n = 3-9 animals/time point). Following PGF(2alpha) administration, TIMP-1 mRNA levels decreased (P < 0.05) at 1 and 2 h relative to 0 h (controls), then increased to levels greater than controls at 4 and 6 h. In contrast, TIMP-2 and -3 mRNA levels did not decrease following PGF(2alpha) administration. The TIMP-1, -2, and -3 proteins were localized to large luteal cells (LLCs) within control (untreated) tissues. However, histodepletion of TIMP-1 within LLCs was evident within 30 min (earliest time point collected) following PGF(2alpha) injection and continued through 48 h. Luteal concentration of TIMP-1, as determined by RIA, was decreased (P < 0.05) by 15 min (earliest time point collected) following PGF(2alpha) administration and remained low through 48 h. In contrast, TIMP-2 and -3 immunolocalization was not altered by PGF(2alpha) administration. Experiment 2 was conducted to determine if PGF(2alpha) could initiate the preceding changes in TIMP-1 in early (Day 3) corpora lutea that can bind PGF(2alpha) but are refractory to its luteolytic effects. Serum concentrations of progesterone and luteal concentrations of TIMP-1 mRNA and protein were similar at 0 and 6 h after PGF(2alpha) injection on Day 3 postestrus. These data suggest that an early and sustained effect of PGF(2alpha) is the specific depletion of TIMP-1 within LLCs that are capable of responding to the luteolytic action of PGF(2alpha). This action may increase the MMP:TIMP-1 ratio, creating an environment that favors extracellular matrix degradation and, thereby, facilitates both functional and structural regression.