Role of the Non-Receptor Tyrosine Kinase ACK2 in EGF Receptor Degradation
Abstract
Epidermal growth factor receptor and members of the ErbBeta family of receptor tyrosine kinases have been implicated in several mitogenic pathway. Regulated growth factor-mediated signaling relies upon a balance between receptor activation, endocytosis, and degradation. As a result, overexpression or mutations altering receptor kinase activity is often sufficient to cause malignant transformation. We are interested in the emerging role of the nonreceptor tyrosine kinase, ACK2, and its substrate, SH3PXl, in regulating ErbBeta family degradation. Establishing a role for ACK2 and SH3PXl in ErbBeta-2 receptor degradation is especially appealing based on the predictive property between receptor overexpression and breast cancer. Currently, we are interested in characterizing the ACK2-SH3PXl interaction and determining the significance of ACK2-dependent phosphorylation of SH3PXl in cells. To address these objectives, we have carried out deletion analysis studies to delineate the region of the phosphorylation on SH3PXl. Based on these studies, we conclude that SH3PXl phosphorylation is lost in the truncation mutant DeltaC339. In parallel site-directed mutagenesis studies, we conclude that all conserved point- mutants of SH3PXl retain tyrosine phosphorylation. Given these findings, we believe that Mass Spectrometry may provide a more sensitive means to identify the ACK2 phosphorylation site(s) on SH3PXl and efforts have been made to generate recombinant forms of ACK2 and SH3PXl. In addition, in vitro kinase screens for inhibitors of ACK2 have been carried out at the high-throughput facility at Merck& Co., Inc. The pyrido-pyrimidine compound from Park Davis (PD158780) has been shown to effectively block ACK2 in vitro and additional experiments will be carried out to determine its activity in vivo. The ability to regulate this phosphorylation event will help to determine the importance of ACK2 activity in receptor endocytosis and degradation.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2005
- Accession Number
- ADA435047
Entities
People
- Carrie Stearns
Organizations
- Cornell University