Abstract: Background and purpose: The Csk binding protein (CBP), is palmitoylated and thereby targeted to lipid rafts, which are membrane microdomains characterized by specific lipid and protein compositions, implicated in various aspects of cancer cell biology. This study aimed to investigate the effects of CBP palmitoylation site mutation on proliferation and apoptosis of cutaneous squamous cell A431, and to explore the molecular mechanisms. Methods: The lentiviral vector of CBP-EGFP fusion protein was constructed. The A431 cell line with CBP palmitoylation site mutation and the A431 cell line with WT-CBP were established by retrovirus transfection. The experiments were divided into 4 groups: Parental group (A431 cells without transfection), Control group (A431 cells transfected with only EGFP negative control virus), WT-CBP group (A431 cells transfected with WT-CBP-EGFP virus), Mutant-CBP group (A431 cells transfected with CBP-EGFP virus carrying mutation of palmitoylation sites). CBP mRNA levels were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and their protein levels were measured by Western blot. The transfection efficiency was detected by flow cytometry (FCM). After additional culture, the proliferation potential of A431 cells was assayed using Cell Counting Kit (CCK-8), and the apoptotic rate was assessed by FCM. The migration and invasion were assessed by wound healing and transwell assays. The protein levels of Csk and Fyn were detected by Western blot. Results: A431 cell line with stable CBP palmitoylation site mutation or WT-CBP was established. Mutant-CBP inhibited proliferation and induced apoptosis in A431 cell lines. However, the proliferation and induced apoptosis of A431 cells mutated in CBP palmitoylation sites did not change significantly (P>0.05). The WT-CBP significantly decreased the healing rate of A431 cells (P<0.001), while the healing rate of A431 cells mutated in CBP palmitoylation sites did not change significantly (P>0.05). WT-CBP significantly decreased the cell migration and invasion abilities (P<0.001). The migration and invasion abilities of A431 cells with CBP palmitoylation site mutation did not change significantly (P>0.05). Western blot results showed that the expression levels of Csk and Fyn proteins in the WT-CBP group were significantly higher than those in the blank Control group and the negative virus Control group (P<0.001), while the expression levels of Csk and Fyn proteins in the Mutant-CBP group did not change significantly compared with the Control groups (P>0.05).). Conclusion: CBP may inhibit the proliferation of A431 cells. CBP with palmitoylation site mutation could affect the inhibitory effect of CBP on the malignant potential of human cutaneous squamous cell carcinoma.

Key words: Adapter protein, Csk-binding protein, Proliferation, Apoptosis, Cutaneous squamous cell carcinoma