Structural Dynamics of Src-Family Kinase Activation

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DESCRIPTION (provided by applicant): Improper or untimely activation of Src-family kinases, key signal transduction molecules, contributes to many cancers and to AIDS progression. Multiple internal regulatory elements cooperate to hold the proteins in an inactive conformation until kinase activity is required. Mutations or binding of other proteins (including HIV Nef) can displace the SH3 and/or SH2 domain(s) from regulatory positions in the inactive conformation, thereby leading to inappropriate activation and abnormal signalling. It is unclear what the conformational changes are during activation. If detailed descriptions of the changes were obtained, a more complete understanding of Src-family kinases would result, and therefore additional strategies to combat disease-related activation may follow. Hydrogen exchange (HX) mass spectrometry (MS) techniques can be used to investigate structural changes in proteins because hydrogen exchange is very sensitive to changes in protein conformation. HXMS offers the promise of working with small amounts of protein, conditions not possible with most previous biophysical analyses of Src-family kinases. To identify conformational changes in Src-family proteins during normal and vitally-induced activation, three specific aims will be accomplished: (1) Define the conformational requirements for Src-family kinase activation. Forms of Hck that model completely inactive and completely active will be separately probed with HXMS and the results compared, thereby revealing regions of Hck that undergo conformational changes during activation. (2) Determine how viral proteins alter the inactive conformation of Src-family kinases. Activation of Hck by HIV Nef and Lck by the Tip protein from Herpesvirus saimiri will be probed with HXMS. Changes in hydrogen exchange when Hck/Lck are bound versus when free are expected to reveal how and where the viral proteins induce activating conformational changes. Regions of protein:protein interaction should suggest new, alternative areas to target therapeutically. (3) Establish if various Src-famity members have unique conformational responses to HIV Nef Because all Src-family kinases are not activated by HIV Nef, HX will be compared for several family members in the presence of HIV Nef to determine how conformation contributes to the "natural resistance" of some family members towards Nef-induced activation. Taken together, these three Aims are expected to provide substantial information about the role of conformational changes in the activation of Src-family kinases, obvious targets in disease prevention.