Interdisciplinary Training

Yong Chi

"Substrate Identification Using Engineered Cyclin-Dependent Kinases"

Phosphorylations are common modifications to cellular proteins and play and important role in virutally 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 the 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 kinase from the large pool for cellular kinases.

The Clurman laboratory has adapted this mutagenesis approach to detect direct 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. 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 roles in cell cycle regulation