Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility

Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p12 5(FAK)) but activate a second member of this kinase family, calcium-depende nt tyrosine kinase (CADTK; also known as Pyk2/CAK beta /RAFTK/FAK2), upon a dhesion or stimulation with chemokines, To study the role of CADTK in monoc yte adherence and motility, we performed immunocytochemical localization th at showed CADTK at the leading edge and fling lamellipodial structures in f reshly isolated, adhered human monocytes, We next introduced CADTK/CAK beta -related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, i nto monocytes by electroporation and showed that it inhibited CADTK autopho sphorylation. Introduction of the fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min after adhesion, (ii) adhesion-induced phosphotyrosine increases and redistribution into lamellipodia, and (iii) adhesion-induced extracell ular signal-regulated protein kinase (ERK) activation. In control experimen ts, introduction of GST or GST-C3 transferase tan inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adh ered in the presence of autologous serum were highly motile even after intr oduction of GST (83% motile cells). However, only 26% of monocytes with int roduced GST-CRNK were motile. In contrast, GST-CRNK-treated monocytes were fully capable of phagocytosis and adhesion-induced cytokine gene induction, suggesting that CADTK is not involved in these cellular activities and tha t GST-CRNK introduction does not inhibit global monocyte functions. These r esults suggest that CADTK is crucial for the in vitro monocyte cytoskeletal reorganization necessary for cell motility and is likely to be required in vivo for recruitment to sites of inflammation.