Current ProjectsProteomics, Breast Cancer, and MLK Signaling PathwaysA survey of human cell lines revealed that the protein levels of MLK3 are much higher in breast cancer cells than in human cell lines derived from other tissues. In order to learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express Flag epitope-tagged MLK3. We are isolating Flag-MLK3 complexes by affinity purification and identifying associated proteins by either in-gel or in-solution trypsin digests followed by tandem MS/MS. These studies are providing new information to understand MLK3 signaling in cancer cells.  Release of Autoinhibition of MLK3 Src homology 3 (SH3) domains typically mediate protein-protein interactions by binding proline-rich sequences in other proteins. Studies in our lab indicate that MLK3 is autoinhibited by an interaction between the N-terminal SH3 of MLK3 and a region that contains a single proline residue that is located between the zipper and the CRIB motif of MLK3. We are using biophysical methods, including analytical ultracentrifugation and fluorescence resonance energy transfer (FRET) to analyze the autoinhibited and activated states of MLK3. In addition were are testing the ability of signaling partners of MLK3 to release the autoinhibition of MLK3 and, hence, activate MLK3.  Phosphorylation
Site Analysis of MLK3 and Other Signaling Proteins
We have been using mass spectrometry as a tool to identify in vivo phosphorylation
sites of MLK3. Phosphospecific antibodies, in combination with site-directed
mutants of MLK3, are being used to evaluate the role of site-specific
phosphorylation on MLK3 activity and signaling. |