Identification of a PTEN mutation with reduced protein stability, phosphatase activity, and nuclear localization in Hong Kong patients with autistic features, neurodevelopmental delays, and macrocephaly

Abstract

PTEN is a tumor suppressor gene inactivated in over 30% of human cancers. It encodes a lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3-kinase signaling pathway. Germline mutation frequently occurs in this gene in patients diagnosed with PTEN Hamartoma Tumor Syndrome (PHTS). PHTS individuals are characterized by macrocephaly, benign growth of multiple tissues and increased tumor risk. In addition, autistic phenotypes are found in 10-20% of individuals carrying the germline PTEN mutation with macrocephaly. In this report, 13 suspected PHTS patients were screened for mutation in the PTEN gene. A missense variant (c. 302T > C) substituting the isoleucine at codon 101 to a threonine, a single nucleotide insertion (c. 327-328insC) causing a frame shift mutation and termination at codon 109, and a nonsense variant (c. 1003C > T) truncated the protein at codon 335 were identified. The I101T mutation significantly reduced PTEN protein expression levels by 2.5- to 4.0-fold. Mechanistically, I101T reduced the protein half-life of PTEN possibly due to enhanced polyubiquitination at Lysine 13. However, the I101T mutant retained almost 30% of the lipid phosphatase activity of the wild-type protein. Finally, the I101T mutant has reduced phosphorylation at a PTEN auto-dephosphorylation site at Threonine 366 and a lowered ratio of nuclear to cytosolic protein level. These partial losses of multiple PTEN biochemical functions may contribute to the tissue overgrowth and autistic features of this PHTS patient. Autism Res 2018, 11: 1098-1109. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. LAY SUMMARY: The genetics of autism spectrum disorders is highly complex with individual risk influenced by both genetic and environmental factors. Mutation in the human PTEN gene confers a high risk of developing autistic behavior. This report revealed that PTEN mutations occurred in 23% of a selected group of Hong Kong patients harboring autistic features with gross overgrowth symptoms. Detailed characterization of a PTEN mutation revealed reduced protein stability as one of the underlying mechanisms responsible for reduced PTEN activity.

Keywords: Hong Kong; PTEN; PTEN hamartoma tumor syndrome; autism spectrum disorders; macrocephaly.

Figure 1

Mutation analysis of PTEN gene. All 9 exons of the human PTEN gene were amplified and subjected to Sanger sequencing analysis. A T to C substitution at position 302 of codon 101 was detected in patient D07018, a C insertion at position 327–328 of codon 109 in patient D02373, and a C to T substitution at position 1003 of codon 335 in patient D09742. Mutation sites are indicated by arrows.

Figure 2

Expression analysis of PTEN I101T mutant. (A) Indicated expression plasmids were transfected in PC3 cells. After 48 hr, cells were solubilized with Laemmli buffer. Western blotting analysis was carried out using anti‐PTEN and anti‐actin antibodies. (B) Similar experiments were conducted in 293T cells with comparable results being obtained. Results were quantified from six independent experiments. Bars, SD. **P < 0.01; Mann–Whitney test. (C) 293T cells were transfected with either WT or I101T expression plasmids. Cells were treated with cycloheximide (80 μg/ml) for the indicated durations. Cells were solubilized in Lammeli buffer and the levels of PTEN were determined by Western blotting analysis and normalized to actin levels. Results from three independent experiments were quantified. Bars, SD. **P < 0.01; Two‐Way ANOVA with Bonferroni posttest.

Figure 3

Enhanced polyubiquitination of PTEN I101T mutant. (A) Western blotting analysis of whole cell extracts of 293T transfected with the indicated expression plasmids. Cells were treated with (+) or without (−) 10 μM of MG132 for 6 hr. The intensity of the polyubiquitinated (PolyUb) PTEN protein species (bracketed) was quantified and normalized to the levels of the unmodified PTEN. Results derived from six independent experiments. Bars, SD. *, P < 0.05; **P < 0.01; ns, not significant; Mann–Whitney test. (B) Western blotting analysis was carried out as outlined in (A) and the relative expression of PTEN was quantified and normalized to actin. Results from six independent experiments. Bars, SD. **P < 0.01; ns, not significant; Mann–Whitney test. (C) Similar lysates were first normalized to give similar PTEN expression and Western blotting analysis was performed using anti‐PTEN antibody to detect the extent of polyubiquitination. The non‐ubiquitinated PTEN protein species are marked with an asterisk.

Figure 4

Phosphorylation state of PTEN I101T mutant. Western blot analysis of 293T cells transfected with WT, ΔCAT and I101T. Similar level of PTEN was immunoprecipitated with an anti‐AU5 antibody and Western blotting analysis was carried out with either an anti‐p‐PTEN S380/T382/T383 (p‐3P) (A) or an anti‐p‐PTEN T366 (B) antibodies. Results were derived from six independent experiments for (A) and seven individual experiments for (B). Bars, SD. **P < 0.01; ***P < 0.001; ns, not significant; Mann–Whitney test.

Figure 5

Lipid phosphatase activity of PTEN I101T mutant. Empty vector control (C), PTEN WT and I101T expression plasmids were transfected in either PC3 (A) or U251 (B) cell lines. The levels of p‐Akt S473 (p‐Akt) and total Akt (c‐Akt) were examined by Western blotting analysis. Specific p‐Akt levels were calculated from six and three individual experiments for PC3 and U251, respectively. Bars, SD. *P < 0.05; **P < 0.01; ns, not significant; Mann–Whitney test. (C) PC3 transfected with indicated plasmids were serum‐starved for overnight and stimulated with 10% FBS for 10 min. The levels of p‐Akt T308, p‐Akt S473 and total Akt (c‐Akt) were examined by Western blotting analysis. Specific p‐Akt levels were calculated from three individual experiments. ns, not significant; Mann–Whitney test. (D) Indicated plasmids were transfected in 293T cells. PTEN was immunoprecipitated by an anti‐AU5 antibody and probed with an anti PTEN antibody (lower panel). PTEN protein levels were normalized and lipid phosphatase assay was carried out with PIP₂+PIP₃ as substrates with released phosphate measured by a Malachite Green Assay kit (Echelon). Lipid phosphatase activities from seven independent experiments were quantified and expressed as pmol ng⁻¹min⁻¹. Bars, SD. **P < 0.01, ***P < 0.001; Mann–Whitney test.

Figure 6

Nuclear localization of PTEN I101T mutant. (A) Immunofluorescence analysis of AU5‐tagged PTEN WT, ΔCAT, and I101T in 293T using an anti‐AU5 antibody and detected with an anti‐mouse IgG antibody conjugated with Alexa 488 fluorophore. Cell nuclei were stained with DAPI. Positive control for nuclear staining was provided by a PTEN tagged with a nuclear localization signal (NLS‐WT). Fluorescent signals were captured by confocal microscopy and representative images are shown. Fluorescent intensity was quantified for the cytosolic and nuclear compartments by ImageJ software and expressed as a relative ratio. Bars, SD; n = 27; ***P < 0.001. ns, not significant. One‐way ANOVA. (B) Live cell imaging of GFP‐PTEN WT and GFP‐PTEN I101T; nuclei were stained with Hoechst 33342. Fluorescent intensity was quantified for the cytosolic and nuclear compartments and expressed as a ratio of the nuclear to cytosol fluorescence intensity. Bars, SD. n = 7, ***P < 0.001. Mann–Whitney test.