Cyclin D1 is an important regulator of cell cycle progression through G1 phase. Although initially considered to be strictly a target of growth factor stimulation, we and now others have shown that the induction of cyclin D 1 actually requires a coordinated signaling by growth factors and cell adhesion to the ECM. Cyclin D1 is typically induced in mid-G1 phase, and this induction requires sustained activation of ERK for approximately 6 h. Cell adhesion to ECM is required for the induction of cyclin D1 because ECM-dependent activation of integrins (at least alpha5beta1 integrin) sustains ERK activity in growth factor-treated cells. Our most recent data show that rho kinase-dependent stress fiber formation is also required for sustained ERK activity and that stress fibers are acting upstream to promote integrin clustering/signaling. We now propose a series of experiments to examine the mechanism by which stress fiber-dependent integrin signaling regulates sustained ERK activity and, in turn, how sustained ERK activity regulates cyclin D1 gene expression.
Aim 1 will define the mechanism by which stress fiber-dependent integrin clustering allows for sustained ERK activity in growth factor-treated MEFs.
Aim 2 will identify the ERK-regulated cis-elements in the cyclin D 1 promoter and the corresponding ERK-regulated transcription factors. We will place particular emphasis on the largely unaddressed question of why the induction of the cyclin D 1 gene is so delayed relative to the rapid activation of ERK and induction of other ERK-dependent genes.
Aim 3 will address the role of PI3K in cyclin D1 gene expression. Like ERK, PI3K is activated by both growth factors and integrin-mediated adhesion, a sustained PI3K signal is required for G1 phase cell cycle progression, and PI3K is required for efficient mid-G1 phase induction of cyclin D1. Therefore using the approaches developed for Aims 1 and 2, we will determine how PI3K regulates the cyclin D1 gene by defining the responsible cis-elements, transcription factors, and PI3K effectors. Finally, we will determine if growth factor receptor tyrosine kinases (RTKs) and integrins cooperate to control the activity of the PI3K effector(s) that regulate G1 phase cyclin D1 gene expression. Together, these experiments will provide us with a comprehensive understanding of the integrated effects of RTKs, integrins, and the actin cytoskeleton on the major signaling pathways responsible for the mid-G1 phase induction of cyclin D1.
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