Fig. 3: daf-18(yh1) substantially decreases lipid phosphatase activity, but partially maintains protein phosphatase activity of DAF-18/PTEN.
a Representative images of rpl-28p::CFP::PHAKT-expressing worms with daf-2(e1370) [daf-2(−)], daf-2(−); daf-18(yh1), or daf-2(−); daf-18(nr2037) [daf-18(−)] mutations (rpl-28p: a promoter of a ubiquitous rpl-28, ribosomal protein large subunit 28). Scale bar: 50 μm. Arrowhead: membrane CFP::PHAKT. b Quantification of membrane-localized PHAKT in worms in panel (a) (n ≥ 23 for each condition, from four independent trials). c, d His-tagged human recombinant PTEN proteins used for in vitro phosphatase assay. WT: wild-type PTEN; C105Y: C105Y mutant PTEN; C124S: C124S mutant PTEN (phosphatase-dead variant). The recombinant proteins were separated by using SDS-PAGE and stained with Coomassie blue (c), and detected by using western blotting with anti-His antibody (d). e, f In vitro PTEN lipid phosphatase assay. Purified recombinant WT, C105Y, and C124S PTEN proteins [127 nM (e) or 42 nM (f)] were incubated with PIP3 substrates (N = 4). g In vitro PTEN protein tyrosine phosphatase assay. Purified recombinant WT, C105Y, and C124S PTEN proteins were incubated with phospho-tyrosine-containing peptides, and the protein phosphatase activities were calculated by detecting free phosphates (N = 9). h Dose-dependent changes in protein phosphatase activities of the PTEN variants (N = 3). See Supplementary Fig. 4f for the comparison of protein phosphatase activities between WT PTEN and protein tyrosine phosphatase 1B (PTP1B), a positive control. Error bars represent the standard error of the mean (s.e.m., ***p < 0.001, n.s.: not significant, two-tailed Student’s t test relative to WT unless otherwise noted). See Source Data for data points used for the derivation of data.
