Research Summary

Yong Chi

Substrate Identification Using Engineered Cyclin-Dependent Kinases

Phosphorylases are common modifications to cellular proteins and play an important role in virtually every cellular process.  Protein kinases represent the largest enzyme family in human cells, but the identification of the direct cellular substrates of individual protein kinases remains the key challenge in this field.  Recently, a novel method for detecting kinase substrates has been developed that introduces an engineered mutation within the kinase ATP-binding pocket.  This allows the mutant kinase to utilize ATP analogs that are excluded by normal kinases, thus isolating the activity of the engineered kinas from the large pool of cellular kinases.

The Clurman laboratory has adapted this mutagenesis approach to detect targets for various G1-specific cyclin-dependent kinases (CDKs).  They have generated several engineered CDKs carrying the relevant ATP-binding domain mutations.  These engineered CDKs can use ATP analogs efficiently, and some no longer use normal ATP.  They have also detected potential kinase substrates in cell lysates using radiolabeled ATP analogs.  However, determination of both the identities of these proteins and their phosphorylation sites requires a tool such as mass spectrometry.  The Aebersold laboratory has played a leading role in method developments in proteomcics and mass spectrometry.  Collaboration between these groups will combine the expertise and tools available to achieve the aims of this study:  to identify novel substrates of human CDKs and study their rolls in cell cycle regulation.  To our knowledge, it is the first time such a novel approach is used to identify CDK substrates.  We hope our study will not only provide new clues to CDK and cell cycle regulation, but will also have broad positive influences on the study of other kinases.

	Dual Mentor Application
	2010 Fellows
	2009 Fellows
	2008 Fellows
	2007 Fellows
	2006 Fellows
	2005 Fellows
	2004 Fellows
	2003 Fellows
	2002 Fellows
	2001 Fellows
	2000 Fellows
	1999 Fellows
	1998 Fellows