Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility.
Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed CADTK at the leading edge and ruffling lamellipodial structures in freshly isolated, adhered human monocytes. We next introduced CADTK/CAKbeta-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and showed that it inhibited CADTK autophosphorylation. 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 extracellular signal-regulated protein kinase (ERK) activation. In control experiments, introduction of GST or GST-C3 transferase (an inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adhered in the presence of autologous serum were highly motile even after introduction of GST (83% motile cells). However, only 26% of monocytes with introduced 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 that GST-CRNK introduction does not inhibit global monocyte functions. These results 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.
Watson
J M
JM
University of North Carolina Lineberger Comprehensive Cancer Center, Department of Medicine and Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.
Harding
T W
TW
Golubovskaya
V
V
Morris
J S
JS
Hunter
D
D
Li
X
X
Haskill
J S
JS
Earp
H S
HS
D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2; D006801:Humans; D066298:In Vitro Techniques; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D010587:Phagocytosis / Q000502:physiology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D020928:Mitogen-Activated Protein Kinases