Abstract
PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway.
Highlights
Introduction(d) PCTK3-knockdown HEK293T cells were serum starved for 6 hours, and were treated with 25 μM Y27632 for 4 hours
We revealed that PCTK3 knockdown increased cell motility in HEK293T cells
We demonstrated that the phosphorylation of myosin light chain (MLC) at Thr-18 and Ser-19 was increased in PCTK3-knockdown cells, and MLC phosphorylation was suppressed by the Rho-associated kinase (ROCK) inhibitor Y27632
Summary
(d) PCTK3-knockdown HEK293T cells were serum starved for 6 hours, and were treated with 25 μM Y27632 for 4 hours. (e,f) PCTK3-knockdown HEK293T cells were serum starved and were treated with Y27632 for 4 hours. G protein-coupled receptor-induced phosphorylation of FAK at Ser-843 downregulates FAK activity[28,29] It has been demonstrated the importance of FAK in the regulation of cell motility, the regulatory mechanism of FAK is not fully understood. We determined that PCTK3 regulates cell migration by controlling the balance between RhoA and Rac[1] activities. We found that PCTK3 suppresses cell adhesion and spreading by negatively modulating the FAK activity. These findings are valuable for future investigations of PCTK3 function
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