Fig 2: FNIP1-D Serine Phosphorylation Gradually Inhibits Hsp90 ATPase Activity(A) FNIP1-D-HA (wild-type [WT]) was overexpressed in HEK293 cells, and the cell lysates were challenged with ATP-agarose for 1 h at 4°C, washed, and then incubated with or without 10 µM of the Hsp90 inhibitor ganetespib (GB) for an additional 1 h at 4°C. ATP-agarose was washed, and ATP-bound Hsp90 was analyzed by immunoblotting. EV was used as control.(B) Non-phosphorylatable FNIP1-D-HA (WT) and its mutants were transiently expressed in HEK293 cells, and cell lysates were incubated with ATP-agarose. Hsp90 binding to ATP was examined by immunoblotting.(C) Schematic representation of creating FNIP1-D phosphomimetic mutants for expression and purification from bacteria. EV was used as control.(D) Inhibitory effect of the recombinant FNIP1-D-His6 phosphomimetic mutants on ATPase activity of Hsp90a-FLAG isolated from PC3 cells was measured in vitro. A Student’s t test was performed to assess statistical significance (n.s., not significant).

Fig 3: O-GlcNAcylation Antagonizes Phosphorylation of FNIP1(A) In vitro O-GlcNAcylation of recombinant FNIP1-D-His6 and its non-phosphomutants. O-GlcNAcylation and interaction of FNIP1-D-His6 and its mutants with OGT-FLAG were assessed by immunoblotting.(B) FNIP1-D-HA and its non-phosphomutants were co-expressed with either EV or OGT-V5 in HEK293 cells. Serine phosphorylation and O-GlcNAcylation of FNIP-1-D-HA and its mutants as well as their interaction with Hsp90 and OGT were assessed by IP and immunoblotting.(C) Schematic representation of O-GlcNAcylation of FNIP1.

Fig 4: Relay Serine Phosphorylation of FNIP1 Gradually Activates Both Kinase and Non-kinase Clients(A) RLM1-LacZ activity was measured in yeast expressing WT hHsp90a and WT FNIP1-FLAG and its non-phosphomutants. EV was used as a control. Cells were grown to mid-log phase and stressed with 8 mM caffeine for 4 h. Data are presented as mean ± SD derived from three independent experiments. A Student’s t test was performed to assess statistical significance (**p < 0.01). FNIP1-FLAG and Hsp90a were visualized by immunoblotting.(B) Yeast with WT hHsp90a, expressing WT FNIP1-FLAG, and its non-phosphomutants were also co-expressing Ste11?N-cMyc under the GAL1 promoter. EV was used as a control. Cells were grown on glucose (-) or galactose (+) media, and Ste11?N-cMyc protein expression was examined by immunoblotting.(C) GAL1-v-SRC was transformed into yeast cells with WT hHsp90a, containing WT FNIP1-FLAG and its non-phosphomutants. EV was used as a control. Cells were grown on glucose (-) or galactose (+) media. v-Src and total phosphotyrosine were detected by immunoblotting.(D) GR-lacZ activity was examined in cells expressing WT hHsp90a and WT FNIP1-FLAG and its non-phosphomutants. EV was used as a control. Data are expressed as a percentage of the activity observed in WT cells and presented as the mean ± SD from values obtained in three independent experiments. A Student’s t test was performed to assess statistical significance (***p < 0.001). FNIP1-FLAG and Hsp90a were visualized by immunoblotting.(E) The above yeast strains expressing HSE-LacZ were heat shocked at 39°C for 40 min. The heat shock response was measured in three independent experiments. All data represent mean ± SD. EV was used as a control. A Student’s t test was performed to assess statistical significance (****p < 0.0001). FNIP1-FLAG and Hsp90a were examined by immunoblotting.(F) CFTR-HA was expressed in the above yeast strains and treated with cycloheximide (CHX; 50 mM) and harvested at the indicated time points. CFTR-HA FNIP1-FLAG and Hsp90a were analyzed by immunoblotting.(G) Quantification and representation of kinase and non-kinase clients stability and/or activity obtained from data in (A)–(F). Results are presented as a heatmap.p values are for Pearson correlation of the trend between number of phospho-sites in each FNIP1 construct and client stability or activity.

Fig 5: O-GlcNAcylation Leads to Ubiquitination of K1119-FNIP1 and Proteasomal Degradation(A) HEK293 cells were transiently transfected with FNIP1-FLAG and S938A mutant. DAPI was used for nuclear staining, and cells were analyzed by immunofluorescence microscopy.(B) FNIP1-D-FLAG and S938A mutant were transiently expressed alone or co-expressed with OGT-V5 or CK2a-HA in HEK293 cells. 100 µM of the OGA inhibitor PUGNAc was used to treat the cells only expressing FNIP1-D-FLAG and S938A mutant overnight. FNIP1-D-FLAG and its mutant were IP, and their serine phosphorylation and O-GlcNAcylation were detected by immunoblotting.(C) Cells in (B) were used in chase experiment by treating them with 50 µM CHX followed by protein extraction at the indicated time points. The half-life of FNIP1-D-FLAG and S938A mutant was analyzed by immunoblotting.(D) Ubiquitination of samples in (B) detected by immunoblotting.(E) FNIP1-D-FLAG and K1119R mutant were transiently expressed alone or co-expressed with OGT-V5 in HEK293 cells. PUGNAc was used in cells only expressing FNIP1-D-FLAG and K1119R mutant. FNIP1-D-FLAG and its mutant were IP, and ubiquitination was detected by immunoblotting.(F) Samples in (E) were treated with 50 µM CHX followed by protein extraction at the indicated time points. The half-life of FNIP1-D-FLAG and K1119R mutant was examined by immunoblotting.(G) Schematic representation of O-GlcNAcylation of FNIP1-S938 followed by ubiquitination of K1119.