Purchase this article with an account.
J.W. Harbour, L. Worley, D. Ma, P. Zhou; Pattern of Phosphorylation of the Rb Protein in Uveal Melanoma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):930.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously showed that the tumor suppressor Rb is phosphorylated in a stepwise manner by cyclin D-cdk4 then cyclin E-cdk2 as cells progress through G1 and S phase (Harbour et al. Cell, 1999). Phosphorylation of specific sites on Rb regulates distinct functions of Rb. The aim of this study was to determine how Rb is phosphorylated in uveal melanoma in order to gain insights into the biology of this eye cancer. Methods: In vitro phosphorylation assays, colony formation assays, flat cell assays, BrDU incorporation, flow cytometry, western blot analysis, and immunohistochemistry (44 archival uveal melanoma specimens) using phospho-specific Rb antibodies for Ser567, Ser780, or Ser807/811. Statistical analysis was performed using Pearson correlation. Results: Rb was phosphorylated in 3 distinct kinetic steps. The first step involved phosphorylation of C-terminal sites, including Ser780 and Ser807/811. The second step involved phosphorylation of further sites in the A-B pocket. The third, and slowest step, involved phosphorylation of Ser567, which disrupted the A-B pocket and inactivated the Rb protein. In physiologic experiments in cell culture, the C-terminal sites regulated cell cycle, differentiation and senescence functions of Rb. However, phosphorylation of Ser567 appeared to trigger an apoptotic checkpoint. By immunohistochemistry, nuclear staining for phosphorylation at Ser780 was positive in 0-4.9% of cells (mean, 0.9%), Ser807/811 phosphorylation was positive in 0-3.1% of cells (mean, 1.1%), and Ser567 phosphorylation was positive in 0-0.7% of cells (mean, 0.1%). Cytoplasmic staining for Ser567 phosphorylation was positive in 0-7% of cells (mean, 1.0%). Cytoplasmic Ser567 staining correlated strongly with increased expression of cyclin D (p=0.01). By western blot analysis, Ser780 was strongly phosphorylated in 4 of 4 melanomas, Ser807/811 was phosphorylated in 3 of 4 melanomas, and Ser567 was only phosphorylated in degraded Rb. Conclusions: Uveal melanoma cells phosphorylate specific sites on Rb that selectively disrupt it's tumor suppressor functions (e.g. cell cycle, differentiation, and senescence). However, most uveal melanoma cells avoid phosphorylation of Ser567, which can trigger an apoptotic response, by keeping cyclin dependent kinase activity below a certain threshold (e.g. by titrating cyclin D levels). This novel finding could potentially be exploited with therapeutic small molecules to induce selective apoptosis in uveal melanoma cells.
This PDF is available to Subscribers Only