Our study focuses on understanding the relationship between the viral protein K15 and human protein phospholipase C γ1 (PLC-γ1) in Kaposi's sarcoma. K15, expressed in infected endothelial cells, plays a vital role in cell proliferation, angiogenesis, and viral reactivation. Our goal is to understand how K15 activates PLC-γ1 and enhances phospholipase activity. We employ biophysical and structural biology techniques, particularly NMR spectroscopy, to explore the structural basis of K15 recruitment to PLC-γ1.
PLC-γ isoenzymes have a gamma-specific array (γSA), consisting of folded domains: a tandem Src homology 2 (SH2) domain, an SH3 domain, and a split pleckstrin homology domain. The position of γSA relative to the active site determines the protein's activation state. In its inactive state, γSA occupies the catalytic site of PLC-γ1. Activation by receptor tyrosine kinases (RTKs), which leads to phosphorylation of Tyr783 in the cSH2-SH3 linker region, induces a large conformational change that exposes the catalytic site.
We propose that K15 binds and activates PLC-γ1 independently of RTKs. To confirm this, we conduct a detailed investigation of the multidomain γSA in both active and K15-bound states. Our focus is on understanding how K15 alters the orientation of γSA domains.
Our results reveal that a phosphorylated peptide derived from K15, containing the Y481EEVL motif (pK15), exhibits high affinity binding to N-terminal and C-terminal SH2 domains of PLC-γ1. Similarly, a phosphopeptide based on the cSH2-SH3 linker sequence (pY783) binds to the C-SH2 domain. Subsequently, we also investigate the binding within the context of the tandem SH2 domain, finding evidence of crosstalk between the SH2 domains through the binding affinities of wild-type and mutant variants (R694L and R586L) to pK15.
These findings suggest allosteric communication between the tandem SH2 domains, mediated by K15 binding to PLC-γ1, resulting in RTK-independent activation of PLC-γ1. Further understanding of this mechanism could establish the K15-PLC-γ1 complex as a promising therapeutic target for treating Kaposi's sarcoma.