Several malignancies, among which is Kaposi’s sarcoma, are associated with infection by the human herpesvirus 8 (HHV-8). The viral protein K15, expressed by infected endothelial cells, promotes proliferation and angiogenesis and is required for efficient viral reactivation from latency, ultimately leading to carcinogenesis. K15 activity has been linked to its capacity to activate the human protein phospholipase C γ1 (PLC-γ1).
PLC enzymes control cellular events such as proliferation, differentiation and chemotaxis. Uniquely among PLC isotypes, PLC-γ isoenzymes bear a set of folded domains, known as the gamma-specific array (γSA), which includes two Src homology 2 (SH2) domains arranged in tandem (tSH2). The position of the γSA with respect to the active site regulates the protein activation state. In absence of stimuli, the γSA blocks the catalytic site of PLC-γ1, keeping the protein in the inactive state. Upon endogenous activation by receptor tyrosine kinases (RTKs), a large conformational change leads to the exposure of the protein catalytic site.
We propose that K15 can bind and activate PLC-γ1 through a mechanism that is independent of RTKs. Through a combination of NMR spectroscopy and biophysical methods, we are conducting a thorough structural investigation of the multidomain γSA in both its active and K15-bound states to understand how K15 influences the relative disposition of the domains therein.
We find that a K15-derived phosphorylated peptide containing the Y481EEVL motif (pK15) binds tightly to both the N-terminal and C-terminal SH2 domains (nSH2, cSH2) of tSH2 in PLC-γ1. Careful analysis of NMR chemical shift perturbations induced by pK15 binding suggests that pK15 binds to cSH2 in an induced-fit manner. The binding affinities of K15 to wild-type tSH2 and to several tSH2 mutants point to a crosstalk mechanism between the SH2 domains.
Our findings reveal a mechanism whereby pK15 binding promotes a rearrangement of the position of cSH2 relative to the protein catalytic core, resulting in the RTK-independent activation of PLC-γ1.