ABSTRACT: The SRC inhibitor PP2 can block CASK nucleus translocation in macrophage under H5N1 infection. Since PP2 is a general inhibitor for Src family kinases (SFKs), which includes nine members (Csk, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk), we aimed to identify the specific member responsible for the nuclear translocation of CASK. First, we examined the expression of SFKs in macrophages (Csk, Fyn, Fgr, and Hck) following H5N1-IAV infection (Fig. 4A). This observation suggests that Hck is the most abundant SFK and may contribute to virus-induced nuclear translocation of CASK. To test this, we generated constitutively active forms of HCK: HCK△513-524 (deletion mutant) and HCK Y499F mutant (YF mutation) [24], then co-transfected them with an EGFP-CASK construct into 293T cells. Immunoprecipitation with anti-GFP mAb, followed by trypsin digestion and LC-MS/MS analysis, revealed that compared to the control group, the phospho/nonphospho ratio at residue Serine 395 (S395) of CASK increased approximately 10-fold (0.008 to 0.072 by HCK△513-524, 0.008 to 0.108 by HCK Y499F), while the other 8 potential phosphorylation sites did not show significant alterations (Table 1). To confirm the phosphorylation of S395 of CASK is required for nuclear translocation, we generated an EGFP-CASK S395A mutant (SA mutation) to prevent CASK phosphorylation. In the absence of HCK Y499F, both the wild type (CASK S395) and mutant (CASK S395A) remained in the cytosol (left two columns, Fig. 4B). In contrast, transfection of HCK Y499F (red color) induced the translocation of wild type CASK (green color) into the nucleus (3rd column from left, Fig. 4B), an effect not observed with the CASK S395A mutant (4th column from left, Fig. 4B). The percentage of nuclear GFP-CASK (60% and 90% in WT and 35% in S395A) resulting from the overexpression of wild type HCK and HCK Y499F is shown in Figure 4C. Thus, we concluded that activated HCK induces the phosphorylation of CASK at Ser395, facilitating its nuclear translocation.
Jing-Ying Huang 