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Overexpression of the serine/threonine kinase GLK/MAP4K3 in human lung cancer is associated with poor prognosis and recurrence, however, the role of GLK in cancer recurrence remains unclear. Here, we report that transgenic GLK promotes tumor metastasis and cell migration through the scaffold protein IQ motif–containing GTPase-activating protein 1(IQGAP1). GLK transgenic mice displayed enhanced distant metastasis. IQGAP1 was identified as a GLK-interacting protein; two proline-rich regions of GLK and the WW domain of IQGAP1 mediated this interaction. GLK and IQGAP1 colocalized at the leading edge including filopodia and lamellipodia of migrating cells. GLK directly phosphorylated IQGAP1 at Ser-480 enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell migration and lung cancer metastasis were abolished by IQGAP1 depletion. Consistently, human NSCLC patient tissues displayed increased phospho-IQGAP1, which correlated with poor survival. Collectively, GLK promotes lung cancer metastasis by binding to, phosphorylating, and activating IQGAP1.Significance:These findings show the critical role of the GLK–IQGAP cascade in cell migration and tumor metastasis, suggesting it as a potential biomarker and therapeutic target for lung cancer recurrence.

Cell tracking in time-lapse microscopy images of primary lung epithelial cells from wild-type (WT), GLK-deficient (KO), and GLK transgenic (Polâ…¡-GLK)) mice. The tracked path of the mGFP-positive cell is shown as the line.

Supplementary Methods, Figures, and Movies - Supplementary File accompanying the manuscript provides additional details regarding the methods of immunoblotting, cell migration and proliferation assays, time-lapse live cell imaging, FRET, and in vitro kinase assays. Additionally, we also provide IHC data of mouse tissues (Fig. S1), cell migration assays using primary lung cells or lung cancer cell lines (Fig. S2-S6), PLA data (Fig. S7), as well as five N-SIM movies (Movies S1-S5).

Video.avi GLK co-localizes with IQGAP1 at filopodia and lamellipodia of a cell (Cell #3).

Cell tracking in time-lapse microscopy images from of lung epithelial cells from littermate wild-type (WT), GLK transgenic (Polâ…¡-GLK), GLK transgenic/IQGAP1-heterozygous (Polâ…¡-GLK;IQGAP1+/- ), and GLK transgenic/IQGAP1 knockout (Polâ…¡-GLK;IQGAP1-/- ) mice. The tracked path of the migration cell is shown as the line.

Video4.avi GLK co-localizes with IQGAP1 at the leading edge of a migrating cell (Cell #4)

Video 5.avi GLK co-localizes with IQGAP1 at the leading edge of a migrating cell (Cell #5).