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p27Kip1 inhibition of GRB2-SOS formation can regulate Ras activation
Stephanie J Moeller1, Elizabeth D Head, Robert J Sheaff
1University of Minnesota Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Abstract:
p27(Kip1) (p27) is often inappropriately downregulated in aggressive human cancers. Although p27 can inhibit cyclin-dependent kinases (CDKs), low p27 does not always correlate with increased CDK activity. Furthermore, cells derived from p27(-/-) mice respond to antimitogens, maintain restriction point control, and do not deregulate CDKs. Thus, disruption of a p27 function other than CDK inhibition may contribute to the disease state. A yeast two-hybrid screen identified growth factor receptor-bound protein 2 (GRB2) as a p27 binding partner. We now demonstrate that p27 can inhibit GRB2 function by blocking its association with the guanine nucleotide exchange factor SOS. Endogenous p27 is rapidly exported from the nucleus to the cytoplasm in response to mitogen stimulation, where it binds GRB2 concomitant with a decrease in GRB2-associated SOS. As predicted, mitogen-stimulated p27(-/-) cells maintained their GRB2-SOS complexes for significantly longer. The Ras/mitogen-activated protein kinase pathway does not appear to be deregulated in cells lacking p27 despite excess GRB2-SOS, suggesting that additional control mechanisms are present. A transient-transfection approach was employed to show that p27 can inhibit Ras activation by targeting GRB2 and further revealed that the CDK and GRB2 inhibitory functions of p27 are separable and distinct. Thus, p27 downregulation may compromise control of Ras, one of the most common oncogenic events in human cancer.
Insights
Downregulation of p27(Kip1) (p27) in cancer may impair Ras pathway control. p27 inhibits growth factor receptor-bound protein 2 (GRB2) function, distinct from its role in cyclin-dependent kinases (CDKs).
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- p27(Kip1) (p27) is frequently downregulated in aggressive human cancers.
- Low p27 levels do not always correlate with increased cyclin-dependent kinase (CDK) activity, suggesting alternative functions.
- p27(-/-) mouse cells exhibit responses to antimitogens and restriction point control, indicating functions beyond CDK inhibition.
Purpose of the Study:
- To investigate novel functions of p27 beyond its known role in CDK inhibition.
- To identify and characterize p27's interaction with growth factor receptor-bound protein 2 (GRB2).
- To elucidate the role of p27 in regulating the Ras/mitogen-activated protein kinase (MAPK) pathway.
Main Methods:
- Yeast two-hybrid screening to identify p27 binding partners.
- Demonstration of p27 inhibiting GRB2 association with the guanine nucleotide exchange factor SOS.
- Analysis of endogenous p27 localization and GRB2-SOS complex dynamics upon mitogen stimulation.
- Transient-transfection assays to assess p27's effect on Ras activation.
Main Results:
- GRB2 was identified as a p27 binding partner.
- p27 inhibits GRB2 function by preventing its association with SOS.
- Mitogen stimulation causes p27 nuclear export and binding to cytoplasmic GRB2, decreasing GRB2-SOS complexes.
- p27(-/-) cells maintain GRB2-SOS complexes longer after mitogen stimulation.
- p27 inhibits Ras activation by targeting GRB2, with separable CDK and GRB2 inhibitory functions.
Conclusions:
- p27(Kip1) possesses a distinct function in inhibiting the GRB2-SOS complex and Ras activation.
- Downregulation of p27 in cancer may compromise the control of the common oncogenic Ras pathway.
- The findings reveal separable roles for p27 in cell cycle control (CDK inhibition) and signal transduction (GRB2 inhibition).