Fig 2: Model showing the interplay between PHLPP1 and RNF41 in regulation of SGT1 at kinetochores. PHLPP1 dephosphorylates SGT1 and maintains stable SGT1 at kinetochores. In the absence of PHLPP1, E3 ligase RNF41 associates with phosphorylated SGT1 and promotes its degradation and thereby limits SGT1 availability at kinetochores.

Fig 3: Functional interplay between PHLPP1 and RNF41 during kinetochore formation. A, HeLa cells expressing SFB-tagged SGT1 were treated with nocodazole, and later cells were collected at the indicated different times post-release. SGT1 interaction with PHLPP1 and RNF41 during different mitotic stages was tested by immunoblotting with respective antibodies after immunoprecipitation (IP) with streptavidin beads. WB, Western blotting. B, soluble and chromatin fractions were isolated by acid extraction, and localization of SGT1, PHLPP1, and RNF41 was detected by using specific antibodies. Histone H3 and tubulin were used as specific fraction markers. C, localization of HEC1 to kinetochores was tested in cells either expressing control shRNA, PHLPP1 shRNA, or co-expressing PHLPP1 and RNF41 shRNA (scale bar, 2 μm). Quantification of cells with defective HEC1 localization is shown on right (n = 50 cells each). **, p < 0.01, Student's t test. D, localization of CENP-E to kinetochores in these cells was tested by immunofluorescence (scale bar, 2 μm). Quantification of cells with defective CENP-E localization is shown on right (n = 50 cells each). ***, p < 0.001, Students's t test.

Fig 4: Dephosphorylation of SGT1 by PHLPP1 is required for its stability. A, in vitro phosphorylated SGT1 was incubated with purified wild type (WT) or inactive D901N mutant of PHLPP1 from HEK293T cells, and the released phosphate was detected by absorbance at 620 nm after adding Malachite Green reagent, (n = 3 independent experiments), p < 0.01. B, bacterially purified GST SGT1 was subjected to in vitro kinase assay using HEK293T cell lysate (CL), and then the phosphorylated proteins were incubated with either bacterially purified MBP-tagged wild-type (WT) PHLPP1, D901N mutant, or PTEN. The phosphorylation status of recombinant SGT1 was detected by immunoblotting with pIMAGO reagent after purifying SGT1 with glutathione-Sepharose beads. Non-phosphorylated SGT1 was used in the 1st lane to show the specificity of pIMAGO reagent in detecting the phosphorylated proteins. C, bacterially purified GST SGT1 and SGT1 4A (S17A/T233A/S249A/S289A) mutants were subjected to in vitro kinase assay using HEK293T cell lysate, and then the phosphorylated proteins were incubated with bacterially purified MBP-tagged PHLPP1. The released phosphate was measured by absorbance at 620 nm after adding Malachite Green reagent (n = 3 independent experiments), p < 0.01. D, HeLa cells expressing either control or PHLPP1 shRNA were transfected with SFB-tagged SGT1 wild type (WT) and SGT1 4A mutant. The phosphorylation status of SGT1 in these cells was detected by immunoblotting with pIMAGO reagent after immunoprecipitating SGT1 with streptavidin beads. E, HEK293T cells were transfected with SFB SGT1 WT and SGT1 4A along with Myc RNF41, and their interaction was tested by immunoblotting with Myc antibody after immunoprecipitation (IP) with streptavidin beads. WB, Western blotting. F, 293T cells co-transfected with SFB SGT1 WT, SGT1 4A, SGT1 4E (S17E/T233E/S249E/S289E), and Myc RNF41 were treated with MG132, and the levels of SGT1 ubiquitination were detected. G, 293T cells expressing SGT1 WT, SGT1 4A, and SGT1 4E were treated with cycloheximide (CHX) after 24 h of transfection, and their protein levels were detected at the indicated times after immunoblotting with anti-FLAG antibody. Normalized data from SGT1/actin derived from the quantification of the blots by using Image Lab software is shown.

Fig 5: PHLPP1 regulates mitotic progression by interacting with SGT1. A, HEK293T cell lysate expressing triple-tagged SFB-PHLPP1 was subjected to immunoprecipitation with either IgG or FLAG antibody, and its interaction with endogenous SGT1 was detected by immunoblotting with SGT1 antibody. B, HEK293T cell lysate expressing SFB-SGT1 along with either Myc-PHLPP1 or Myc-PTEN was subjected to immunoprecipitation (IP) with SBP beads, and their interaction was detected by immunoblotting with Myc antibody. Normalized data from IP/total protein derived from the quantification of the blots by using Image Lab software are shown. WB, Western blotting. C, PHLPP1 was depleted in HeLa cells by using shRNA. D, transition of cells through mitosis was analyzed by live cell time-lapse microscopy after synchronizing cells using double thymidine block. Time taken by each cell from mitotic entry to separation of cells after cytokinesis was calculated, and the data were plotted for control and PHLPP1-depleted cells (n = 50), p < 0.05. E, U2OS cells stably expressing H2B-mCherry were analyzed by live cell time-lapse microscopy. Time spent by each cell in different stages of mitosis was calculated, and the data were plotted for control and PHLPP1-depleted cells (n = 20). ns, not significant. *, p < 0.05, Student's t test.