INBRE

3T3L1 cells are a well-studied model system for the differentiation of adipocytes. When they reach confluence, they enter a quiescent state. Treatment with insulin, dexamethasone and IBMX (IDX) causes the quiescent cells to re-enter the cell cycle for 1 or 2 final rounds of division and then permanently exit from the cell cycle as they express the adipocyte phenotype.

Quiescent 3T3L1 cells express high levels of the Rb-related protein p130 but little if any p107. Over the first 24 to 48 hours after IDX treatment the expression of p107 increases to a high level while that of p130 drops to a low level. By 4 days after IDX stimulation, p107 and p130 expression return to their former levels. Over this time, Rb protein levels do not show a dramatic change. Data with protein kinase inhibitors suggest that the rapid induction of p107 is critical for differentiation but is not necessary for proliferation.

While members of the Rb family appear to have some functions in common, they show specificity in their functions that appear to be due to specific interactions with a variety of proteins. I would like to ask what specific protein interactions allow p107 to play its specific role in the differentiation of preadipocytes. Most interactions between Rb family members and other proteins have been identified by pair-wise analysis. While this approach has yielded some data, it fails to probe for all of the components of a protein complex. I would like to take a more general approach by combining biochemistry with mass spectrometry. Nuclear extracts from IDX-treated cells will be fractionated by size and by using affinity reagents to separate specific p107 complexes present after IDX treatment. The components of these complexes will be identified by mass spectrometry. These components will be targeted for disruption to determine their role in the differentiation of 3T3L1 cells.

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Updated 10/31/2005

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