Abstract 542: Syk inhibits the activity of protein kinase A by phosphorylating tyrosine 330 of the catalytic subunit in breast cancer cells.
Authors: Yu Shuai , Huang He , Iliuk Anton , Wang Wen-Horng , Tao Weiguo Andy , Post Carol B. , Geahlen Robert L. 
Published: August 2014

Syk is a nonreceptor protein tyrosine kinase with enigmatic roles in tumorigenesis. Although reported to be a tumor suppressor, Syk also protects breast cancer cells from oxidative stress induced apoptosis. In many cancers including subsets of adult myeloid leukemia, chronic lymphocytic leukemia, diffuse large B cell lymphoma, and non-Hodgkin lymphoma, Syk acts as a proto-oncogene by promoting cell survival. This has led to trials of Syk inhibitors as therapeutic agents for the treatment of hematopoietic malignancies. To identify candidate substrates of Syk that participate in cell survival and apoptotic pathways, we carried out a series of mass spectrometry-based phosphoproteomic screens in cancer cells. In these screens, we found that protein kinase A (PKA) was phosphorylated on a tyrosine (Y330) in the C-terminal tail of the catalytic subunit (PKAc). PKA is regulated, in part, by cis-elements located in its C-terminal tail. One of these elements, the active site tether (AST), acts as a gate for nucleotide entry and egress and is conserved in sequence and function among members of the AGC kinase family. Y330 within the AST is important for recognition of both the nucleotide and peptide substrates and for optimal catalytic efficiency. Interestingly, the other AGC kinases have a phenylalanine instead of a tyrosine at the analogous location.

In this study, we identified and characterized PKAc as a substrate of Syk. Syk catalyzes the phosphorylation of PKAc on Y330 in vitro. Molecular dynamics simulations suggest that, in the unphosphorylated state, the phenyl ring of Y330 is tightly packed against the cavity formed by the β-1,2 strands, the nucleotide, and the flanking residues of AST. In contrast, the phosphorylated tyrosine side chain orients perpendicular to the protein surface, resulting in an increased time-averaged solvent accessible surface area. Indeed, we found that the phosphorylation of PKAc on Y330 by Syk or the replacement of Y330 with glutamate strongly inhibits its catalytic activity. Phosphoproteomic analyses and Western blotting studies indicate that Y330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells. The phosphorylation of a downstream substrate of PKAc, CREB, is inhibited in cells expressing Syk, but can be rescued by a selective inhibitor of Syk. Thus, PKA is a novel substrate of Syk and its phosphorylation on Y330 inhibits its participation in downstream signaling pathways. This study reveals a new mechanism by which Syk can regulate signaling pathways in cancer cells.

Citation Format: Shuai Yu, He Huang, Anton Iliuk, Wen-Horng Wang, Weiguo Andy Tao, Carol B. Post, Robert L. Geahlen. Syk inhibits the activity of protein kinase A by phosphorylating tyrosine 330 of the catalytic subunit in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 542. doi:10.1158/1538-7445.AM2013-542