KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma‐associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV‐TK, in contrast to HSV1‐TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto‐phosphorylation of tyrosines 65, 85 and 120 in the N‐terminus of KSHV‐TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3‐Kinase, respectively. The interaction of Crk with KSHV‐TK leads to tyrosine phoshorylation of this cellular adaptor. Auto‐phosphorylation of KSHV‐TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA‐ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV‐TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV‐TK modulates signalling and cell morphology. Synopsis Gammaherpesviruses are known to encode very inefficient thymidine kinases. KSHV thymidine kinase is actually a tyrosine kinase that induces focal adhesion disassembly via its interaction with known regulators including members of the Crk‐related family of adaptor proteins. KSHV‐TK has tyrosine kinase activity and is autophosphorylated on three key tyrosine residues present within its unique N‐terminal domain, namely residues Y65, Y85 and Y120. Phosphorylated KSHV‐TK suppresses paxillin phosphorylation, induces cell contraction, and promotes the disassembly of focal adhesions via a RhoA‐ROCK‐Myosin II‐dependent pathway. Phospho‐Y65 and ‐Y85 bind to the SH2 domain of CrkI, II and L, whereas phospho‐Y120 is shown to bind to the p85 subunit of PI3‐Kinase. The focal adhesion disassembly induced by KSHV‐TK can be blocked by co‐expression of the SH2 domains of CrkI/II and L, and by drugs or dominant negative mutants that block the RhoA‐ROCK‐Myosin II signalling pathways. Graphical Abstract Gammaherpesviruses are known to encode very inefficient thymidine kinases. KSHV thymidine kinase is actually a tyrosine kinase that induces focal adhesion disassembly via its interaction with known regulators including members of the Crk‐related family of adaptor proteins.