ABSTRACT Heterozygous germline mutations in PTEN gene predispose to hamartomas and tumors in different tissues, as well as to neurodevelopmental disorders, and define at genetic level the

PTEN Hamartoma Tumor Syndrome (PHTS). The major physiologic role of PTEN protein is the dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), counteracting the

pro-oncogenic function of phosphatidylinositol 3-kinase (PI3K), and PTEN mutations in PHTS patients frequently abrogate PTEN PIP3 catalytic activity. PTEN also displays non-canonical

PIP3-independent functions, but their involvement in PHTS pathogeny is less understood. We have previously identified and described, at clinical and genetic level, novel PTEN variants of

cell expression levels and protein stability, PIP3-phosphatase activity, and subcellular localization. In addition, caspase-3 cleavage analysis in cells has been assessed using a C2-domain

caspase-3 cleavage-specific anti-PTEN antibody. We have found complex patterns of functional activity on PTEN variants, ranging from loss of PIP3-phosphatase activity, diminished protein

expression and stability, and altered nuclear/cytoplasmic localization, to intact functional properties, when compared with PTEN wild type. Furthermore, we have found that PTEN cleavage at

the C2-domain by the pro-apoptotic protease caspase-3 is diminished in specific PTEN PHTS variants. Our findings illustrate the multifaceted molecular features of pathogenic PTEN protein

variants, which could account for the complexity in the genotype/phenotype manifestations of PHTS patients. You have full access to this article via your institution. Download PDF SIMILAR

CONTENT BEING VIEWED BY OTHERS DIFFERENTIAL CELL CYCLE CHECKPOINT EVASION BY _PTEN_ GERMLINE MUTATIONS ASSOCIATED WITH DICHOTOMOUS PHENOTYPES OF CANCER VERSUS AUTISM SPECTRUM DISORDER

Article 01 November 2023 PATHOGENIC VARIANTS IN _PIDD1_ LEAD TO AN AUTOSOMAL RECESSIVE NEURODEVELOPMENTAL DISORDER WITH PACHYGYRIA AND PSYCHIATRIC FEATURES Article Open access 24 June 2021

PATHOGENIC VARIANTS CAUSING _ABL1_ MALFORMATION SYNDROME CLUSTER IN A MYRISTOYL-BINDING POCKET AND INCREASE TYROSINE KINASE ACTIVITY Article Open access 22 November 2020 INTRODUCTION

Heterozygous germline mutations in the tumor suppressor gene _PTEN_ (MIM# 601728) define at genetic level the PTEN Hamartoma Tumor Syndrome (PHTS), a complex group of diseases which display

highly heterogeneous phenotypes in carrier patients, from severe malformations and high cancer risk to mild neurodevelopmental manifestations. Major syndromes within PHTS include Cowden

Syndrome (CS), Bannayan-Riley-Ruvalcaba Syndrome (BRRS), Lhermitte-Duclos Disease (LDD), and Proteus-like Syndrome (MIM# 158350), as well as PTEN autism spectrum disorder (PTEN-ASD)

(MIM#605309) [1]. In addition, mutations or absence of the _PTEN_ gene, as well as decrease in PTEN protein expression, are frequent in sporadic tumors from a variety of cancers [2, 3].

_PTEN_ is a unique gene encoding a major PTEN protein of 403 amino acids with phosphatase activity, which counteracts the pro-oncogenic action of class I phosphatidylinositol 3-kinases

(PI3K) by converting phosphatidylinositol 3,4,5-trisphosphate (PIP3) in phosphatidylinositol 4,5-bisphosphate (PIP2) [4, 5]. In addition to its PIP3-phosphatase canonical function, PTEN also

exerts non-canonical functions as a protein phosphatase and as a catalytically-independent cell homeostasis regulator [6]. A major aspect of PTEN function regulation involves the dynamic

changes on its subcellular localization under variable cell conditions, with an active and tightly regulated shuttling between different cellular compartments [7]. In particular, the entry

and accumulation of PTEN in the nucleus affects its accessibility to PIP3 substrate at the cell membranes, and makes PTEN competent to perform nuclear phosphatase-independent activities that

regulate essential processes including chromosome stability, DNA repair, and gene transcription, among others [8]. Multiple layers of regulation of PTEN biological activity exist, from

transcriptional and post-transcriptional regulation of _PTEN_ gene expression to regulation of PTEN translation and reversible post-translational modifications [4, 9, 10]. PTEN is

non-reversibly cleaved by caspase-3 during apoptosis. This cleavage targets the PTEN C-terminal tail in a PTEN-phosphorylation-dependent manner, as well as the Asp301 residue at the PTEN

C2-domain independently of C-tail phosphorylation [11]. PTEN cleavage regulates PTEN protein half-life and the opening of PTEN to its active conformation competent to bind to membranes [12].

In addition, PTEN caspase cleavage in response to chemotherapy it has been proposed as a potential therapy-resistance mechanism [13]. PTEN intrinsic protein stability is highly dependent on

intramolecular interactions in the PTP/C2 domain interface [14], and it is further regulated during cell signaling by phosphorylation, ubiquitination, and protein-interaction events

[15,16,17]. Due to this multi-layered regulation of PTEN protein functions, _PTEN_ missense mutations associated to disease not only target PTEN catalytic residues but also affect residues

essential for protein stability, subcellular localization, and protein-protein interactions. This makes difficult, in many cases, the prediction of the functional properties of the PTEN

variants present in patients, making necessary to perform dedicated, mutation-specific functional studies. Using mammalian and yeast cell models as high-throughput platforms, several global

analyses of function and protein stability of PTEN variants have been performed, which provide an invaluable guide for experimental research and for the clinics [18, 19]. In addition,

dedicated studies on selected panels of mutations associated to disease have unveiled that germline PTEN variants associated to ASD display, in general, higher levels of PIP3-phosphatase

activity in cells than PTEN variants associated to tumor syndromes. Alterations in nuclear/cytoplasmic localization, as well as diminished protein stability, have also been found frequently

in ASD-associated PTEN variants [20,21,22]. A multi-model analysis of functional properties of PTEN variants also highlighted the recurrence of decreased PTEN protein stability, although

distinct molecular mechanisms of PTEN dysfunction could not be related to distinct pathogenic phenotypes [23]. Other experimental cell and animal models, as well as in silico studies, have

been reported that complement the available resources on PTEN mutation-function relationship [24,25,26]. However, in many cases it is still necessary to analyse individually the functional

properties of the distinct PTEN variants found in patients if precision PHTS therapies are aimed to be applied in the next future [27, 28]. We have previously reported the clinical

characteristics of a large group of Spanish PHTS patients, including carriers of _PTEN_ mutations encoding novel PTEN variants of unknown functional significance [29]. Here, we present the

functional characterization of these PTEN variants using a panel of functional, biochemical, and immunochemical assays. Our results illustrate the complexity of the functional phenotypes

associated to some PTEN variants, including the lack of apparent functional alterations using in vitro experimental settings. In addition, we demonstrate for the first time variant-specific

alterations in PTEN cleavage at the C2-domain unstructured loop by the pro-apoptotic protease caspase-3. MATERIALS AND METHODS CELLS AND TRANSFECTIONS Simian kidney COS-7 cells, human breast

carcinoma MCF-7 cells, and HEK293 human kidney carcinoma were grown at 37 °C, 5% CO2 in DMEM containing high glucose supplemented with 5% (COS-7) or 10% (MCF-7, HEK293) heat-inactivated

fetal bovine serum (FBS), 1  mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Human U87MG glioblastoma cells were grown in DMEM containing high glucose supplemented with 10%

heat-inactivated fetal bovine serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The _Saccharomyces

cerevisiae_ strain YPH499 (_MAT_a _ade2-101 trp1-63 leu2-1 ura3-52 his3-_Δ_200 lys2-801_) was used for heterologous expression of mammalian proteins. YPH499 yeast cells were grown in

synthetic complete (SC) medium, containing 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate supplemented with appropriate amino acids and nucleic acid bases, and added 2%

glucose (SD), galactose (SG) or raffinose (SR), as required. COS-7 cells were transfected using GenJet reagent (SignaGen, USA) according to the manufacturer instructions, and processed for

analysis after 48 h. PLASMIDS, MUTAGENESIS, AND VARIANT INFORMATION The pRK5 PTEN, pYES2 PTEN, pRK5 GST-PTEN, YCpLG myc-p110α-CAAX, and pSG5 AKT1 plasmids have been described [20, 30,31,32].

The PTEN and GST-PTEN amino acid substitution and truncation variants were made by PCR oligonucleotide site-directed mutagenesis as described [33], and mutations were confirmed by DNA

sequencing. Nucleotide and amino acid numbering for PTEN variants correspond to reference sequences from accession numbers NM_000314 and NP_000305, respectively. Nomenclature of variants is

according to HGVS. Variants and phenotypes data have been deposited in the LOVD gene variant database (http://www.lovd.nl/3.0/home), with the following accession IDs: 0000352584 (T26I),

0000878862 (P95R), 0000878883 (Y177N), 0000878885 (Q261E), 0000878886 (T277A), 0000878890 (D310G). PROTEIN STABILITY AND PHOSPHATASE ACTIVITY EXPERIMENTS Whole cell protein extracts from

COS-7 cells overexpressing ectopic PTEN variants were prepared by cell lysis in ice-cold M-PERTM lysis buffer (ThermoFisher Scientific) supplemented with PhosSTOP phosphatase inhibitor and

cOmplete protease inhibitor cocktails (Roche, Switzerland), followed by centrifugation at 15200 _g_ for 10 min and collection of the supernatant. Proteins (50–100 μg) were resolved in 10%

SDS-PAGE under reducing conditions and transferred to PVDF membranes for immunoblot analysis. For experiments of protein stability, cells were treated with 800 μg/mL cycloheximide (CHX)

(Sigma Aldrich) or with 10 μM proteasome inhibitor MG132 (Sigma Aldrich) for 6 h before lysis. Immunoblotting was performed using anti-PTEN 6H2.1 monoclonal antibody (mAb) (Merck Millipore,

USA) and anti-GAPDH (Santa Cruz Biotechnology, USA) antibody, followed by IRDye-conjugated anti-rabbit or anti-mouse (LI-COR, USA) antibodies. For experiments of PTEN phosphatase activity in

mammalian cells, COS-7 cells were co-transfected with the appropriate PTEN variants and HA-AKT1, and lysates were processed for immunoblot using anti-phospho-Ser473-AKT, anti-AKT (Cell

Signaling Technologies, USA), and anti-PTEN 6H2.1 antibodies. For determination of phospho-AKT content and PTEN protein stability, bands were visualized and quantified using an Image

studioTM software with Odyssey® CLx Imaging System (LI-COR). For experiments of PTEN phosphatase activity in _S. cerevisiae_, yeasts were transformed by standard procedures, and drop growth

assays and GFP-AKT1 distribution analyses were performed as described [30, 34, 35]. MRNA ISOLATION AND RT-QPCR Real time-quantitative PCR (RT-qPCR) was performed using RNA from COS-7 cells

transfected with the plasmids of interest. Total RNA was isolated using the IllustraRNAspin mini purification kit (GE Healthcare Life Sciences). 1 μg of total RNA was used for cDNA synthesis

using RevertAidTM reverse transcriptase protocol (ThermoFisher Scientific), oligo (dT)18 primers, and RiboLock and RNase inhibitor (all from Fermentas). qPCR was performed as previously

described [36], using Agilent AriaMx Real-Time PCR System (Agilent Technologies) and PTEN validated and reference gene (hypoxanthine phosphoribosyltransferase 1 [HPRT1]) QuantiTect primers

(Qiagen). Relative quantification was performed using the comparative ΔΔCt method. IMMUNOFLUORESCENCE AND MICROSCOPY TECHNIQUES PTEN subcellular location in COS-7 cells was determined by

standard immunofluorescence as previously described, using anti-PTEN 425 A mAb and fluorescein-conjugated anti-mouse antibody [31, 37]. For quantification of PTEN nuclear/cytoplasmic

distribution, at least 50 positive cells were scored for each experiment. Cells were rated as showing nuclear staining (N), cytoplasmic staining (C), or staining within both the nucleus and

the cytoplasm (N/C). Representative examples of the distinct subcellular localizations determined in our scoring are provided in this section. Nuclei were identified by Hoechst

(Sigma-Aldrich) staining. All pictures were taken under a 20X magnification. Measurement of GFP-AKT1 plasma membrane localization in yeast, as an indirect indicator of cellular PIP3 levels,

was performed by fluorescence microscopy, as described [30, 34, 35]. ≥100 cells were examined and scored for each condition or experiment for either cytoplasmic or membrane-associated

localization. CASPASE-3 CLEAVAGE-SITE EXPERIMENTS Whole cell protein extracts from COS-7 cells overexpressing ectopic GST-PTEN or PTEN variants were prepared and resolved by 10% or 12%

SDS-PAGE as indicated above. In the case of PTEN overexpression, cells were kept untreated or were treated with 50 ng/ml TNF-α (Sigma Aldrich). Immunoblotting was performed to monitor PTEN

cleavage at Asp301, using anti-PTEN SP227 mAb (Sigma Aldrich). Anti-PTEN 6H2.1 mAb (Merck Millipore) and anti-GST antibody [38] were used as controls. IRDye-conjugated anti-rabbit or

anti-mouse antibodies (LI-COR) were used as secondary antibodies. RESULTS PTEN GERMLINE VARIANTS OF UNKNOWN FUNCTION Our previous clinical and genetic analysis from a cohort of PHTS patients

revealed several cases carrying PTEN germline variants, in some cases not previously described, which were considered of unknown significance [29]. These included the PTEN variants T26I

[c.77 C > T, p.(Thr26Ile)], P95R [c.284 C > G, p.(Pro95Arg)], Y177N [c.529 T > A, p.(Tyr177Asn)], Q261E [c.781 C > G, p.(Gln261Glu)], T277A [c.829 A > G, p.(Thr277Ala)], and

D310G [c.929 A > G, p.(Asp310Gly)]. Variants Y177N and Q261E have not been annotated, up to date, in sporadic tumor mutation databases (Table 1). Figure 1 illustrates, in the

three-dimensional structure of PTEN protein, the localization of the amino acids targeted by mutations causing these variants. As shown, targeted residues are located on both the PTP- and

the C2-domain of PTEN. T26I variant targets the N-terminal nuclear localization signal (NLS) of PTEN, and it has been partially characterized by us [20, 31], whereas P95R variant targets the

WPD catalytic loop [14]. The rest of variants do not target amino acids manifestly involved in PTEN catalysis or subcellular localization, although residues Y177 and T277 are located at the

PTEN PTP/C2 domain interface, which is important to preserve PTEN functional conformation [14]. The phenotype of the carrier patients is heterogeneous, while they have in common

macrocephaly as a referred manifestation, and ASD features were also frequent. Of interest, only one patient (D310G carrier) displayed clear cancer manifestations. This is coincident with

the finding of D310G mutation in sporadic tumor samples, as annotated in the databases (Table 1; [29]). The D310G variant targets the end of the unstructured loop (residues 286-309) in the

C2-domain of PTEN [14] (Fig. 1), whose physiologic function remains unknown. Since determination of the functional properties of the PTEN proteins encoded by these variants may provide

insights into their potential pathogenicity, including risk for cancer, a broad PTEN functional and immunochemical analysis was performed on cells ectopically expressing the variants.

EXPRESSION, PROTEIN STABILITY AND DEGRADATION OF PTEN PHTS VARIANTS The cellular expression levels of PTEN protein are determinant for PHTS pathogenesis, and decreased expression or low

protein stability of PTEN is relatively frequent in PTEN PHTS variants [22, 23, 30]. The monitoring of the steady-state expression levels of the PTEN variants under scrutiny reflected lower

expression of the Y177N and T277A variants compared to PTEN wild type, whereas the rest of variants displayed expression levels similar than PTEN wild type. The PTEN PHTS variant D252G

[c.755 A > G; p.(Asp252Gly)], previously reported as unstable [22], also displayed lower steady-state expression levels. This expression pattern was consistently observed in several cell

lines, including COS-7 (monkey kidney adenocarcinoma), MCF-7 (human breast carcinoma), U87MG (human glioblastoma), and HEK-293 (human kidney adenocarcinoma) (Fig. 2A, B). To rule out the

possibility of distinct expression levels of the mRNAs encoding the distinct PTEN variants in our transfection experiments, we performed parallel qPCR experiments, which showed no

significant differences in the mRNA levels of the variants under our experimental conditions (Fig. 2C). These results suggest a shorter half-live for Y177N and T277A PTEN variants than PTEN

wild type. PTEN degradation is exerted in part by the ubiquitin-proteasome pathway [39, 40]. Thus, we performed experiments in the presence of cycloheximide or MG132, to test for the

abundance of these variants after protein synthesis- or proteasome-inhibition, respectively, in comparison with PTEN wild type or PTEN D252G. As shown, the variants Y177N, T277A, and D252G

displayed a decrease in their protein abundance after cycloheximide cell treatment, when compared to PTEN wild type or the other PTEN variants (Fig. 2D). In the presence of the proteasome

inhibitor MG132, the Y177N, T277A, and D252G variants, and to a less extent the D310G variant, showed a more prominent relative accumulation than PTEN wild type (Fig. 2E), suggesting that

their decrease in expression is due, at least in part, to increased proteasome-mediated degradation. As mentioned, both Y177 and T277 are located at the PTP/C2 domain interface of PTEN, and

T277 forms hydrogen bonds with amino acids V191 and G251, which would be lost in the T277A variant (https://www3.cmbi.umcn.nl/hope/input/). Thus, the PTEN PHTS protein variants Y177N and

T277A show diminished expression levels in cells, likely due to altered conformation and decreased protein stability, and increased proteasome-mediated degradation. PIP3 PHOSPHATASE ACTIVITY

IN CELLS OF PTEN PHTS VARIANTS To test the activity of PTEN in mammalian cells, we determined the phosphorylation of AKT, as a surrogate marker of PTEN PIP3 phosphatase activity, in the

presence of the different PTEN variants. COS-7 cells were co-transfected with plasmids encoding the PTEN variants and AKT1, and pAKT content was monitored by immunoblot using an anti-pAKT

(Ser473) antibody. PTEN wild type (WT) and the catalytically inactive PTEN C124S [c.371 G > C; p.(Cys124Ser)] mutation were used for comparisons. In this assay, basal pAKT content in

cells is detected upon transfection with empy vector (EV), and pAKT levels diminish upon transfection of PTEN WT, but not upon transfection of catalytically defective PTEN variants. As

shown, the P95R variant was fully inactive, the T26I and Y177N variants displayed decreased activity, and the Q261E, T277A, and D310G variants displayed similar activity than PTEN wild type

(Fig. 3A). Next, we tested the activity of the PTEN variants using our heterologous yeast system, which monitors with high fidelity and sensitivity the PIP3 phosphatase activity of PTEN in

eukaryotic cells, by measuring PIP3 levels in the membrane of the yeast upon ectopic expression of mammalian hyperactive PI3K catalytic subunit (p110α-CAAX) and PTEN [30, 34]. The results of

PTEN activity from the yeast were concordant with the results from mammalian cells, with a PIP3 phosphatase activity as follows: Q261E,T277A,D310G > T26I,Y177N > P95R (Fig. 3B). We

conclude from our determinations that P95R variant lacks PIP3 phosphatase activity in cells, T26I and Y177N variants display compromised PIP3 phosphatase activity, and Q261E, T277A and D310G

variants display PIP3 phosphatase activity similar than PTEN wild type (Fig. 3C).This is of interest in the case of T277A, since this variant displayed low steady-state expression levels

compared to PTEN wild type (Fig. 2). In this regard, Yang et al. studied the PTEN T277A variant in the context of sporadic brain cancer, reporting a decreased half-live for it, in

association with a slight decrease in PIP3 phosphatase activity in cells [41]. Although slight changes in PTEN PIP3 phosphatase activity may account for pathogenicity, the possibility exists

that additional deleterious mechanisms of PTEN function exist in variants such as PTEN T277A. SUBCELLULAR LOCALIZATION OF PTEN PHTS VARIANTS PTEN protein is known to actively shuttle

between cytosolic and nuclear compartments by different mechanisms, which constitutes an important determinant of PTEN biological functions [31, 42]. This prompted us to examine the

subcellular localization of the PTEN variants by immunofluorescence analysis, using anti-PTEN antibodies, both in a PTEN wild type background (which mostly locates in the cytoplasm) and on a

PTEN 1-375 background (which accumulates in the nucleus) [43]. The PTEN variants analysed did not show major differences in the subcellular localization with respect to PTEN wild type,

except for the P95R, Y177N, and T277A variants, which displayed a slight shift towards more marked cytoplasmic localization (Fig. 4A). Remarkably, the nuclear accumulation of PTEN 1-375 was

partially prevented in the Y177N, and T277A variants, whereas the nuclear accumulation of PTEN 1-375 T26I, Q261E, and D310G variants was unaffected. PTEN 1-375 P95R displayed an intermediate

nuclear and nuclear/cytoplasmic localization (Fig. 4B, C). Prevention of nuclear accumulation of PTEN T277A variant in a background of non-C-terminal phosphorylated PTEN-GFP has also been

reported [41]. Together, these findings attribute differential changes in subcellular localization, affected by the PTEN amino acid sequence background, to the P95R, Y177N, and T277A PTEN

PHTS variants. C2-DOMAIN CASPASE-3 CLEAVAGE OF PTEN PHTS VARIANTS PTEN is cleaved by caspase-3 at residue Asp301 (D301) in the unstructured loop from PTEN C2-domain [11] (Figs. 1 and 5A).

This can be readily monitored using the anti-PTEN SP227 mAb, which recognizes the PTEN truncation 1-301 but not PTEN wild type or other PTEN truncations (Fig. 5B). As the use of this mAb for

academic purposes has not been reported yet, we aimed to characterize its epitope specificity for its application in the monitoring of PTEN cleavage at Asp301, an aspect of PTEN biology

with potential functional implications that remains unexplored. Mutation to Ala of residues Gln298 [p.(Gln298Ala), Q298A], Glu299 [p.(Glu299Ala), E299A], Ile300 [p.(Ile300Ala), I300A], or

Asp301 [p.(Asp301Ala), D301A], but not mutation to Ala of residue Asp297 [p.(Asp297Ala), D297A), abrogated the reactivity of SP227 mAb (Fig. 5B). Furthermore, a GST-fusion protein containing

at its C-terminus PTEN residues (298)QEID(301) (GST-QEID) was recognized by SP227 mAb, but not a GST-fusion containing PTEN residues (299)EID(301) (GST-EID) (Fig. 5C). Thus, SP227 mAb

recognizes the C-terminal motif -QEID generated on PTEN upon caspase-3 cleavage. PTEN cleavage at Asp301 is enhanced upon apoptotic stimulation, such as TNF-α cell treatment [11], making

possible a PTEN functional relevance for this specific modification. However, how PTEN PHTS mutations may affect Asp301 PTEN cleavage is unknown. Next, we monitored the effect of the

analysed PTEN mutations on PTEN Asp301 cleavage. As shown, mutations T26I, P95R, Y177N, and T277A displayed diminished PTEN Asp301 cleavage, although it should be mentioned that the

diminished basal expression of the Y177N and T277A variants hampers the interpretation of the results with these mutations. The catalytically inactive C124S variant also displayed a slight

diminished Asp301 cleavage, especially manifested in the presence of TNF-α. On the other hand, Asp301 cleavage of mutations Q261E and D310G was achieved efficiently, both in the absence and

in the presence of TNF-α (Fig. 5D). We conclude that specific PTEN PHTS variants may have compromised their C2-domain-loop cleavage in cells by caspase-3. DISCUSSION Our functional analysis

of the panel of PTEN PHTS mutations reflects the functional heterogeneity attributed to PTEN variants found in association with disease, with most of the variants analyzed (T26I, P95R,

Y177N, and T277A) manifesting several alterations in our assays (Table 2). This is in accordance with previous reports analysing in parallel other sets of PTEN germline mutations using

complementary methodologies [20, 22, 23, 44], as well as with the comparative analysis of the tumor spectrum phenotype from several _Pten_ knock-in mice [45]. In line with the importance of

PTEN protein expression levels for appropriate PTEN function, we have found two PTEN variants (Y177N, T277A) displaying low steady-state expression and a trend of increased

proteasome-mediated degradation. Dedicated studies are required to ascertain the contribution of altered phosphorylation and ubiquitination of these variants in their altered protein

stability. In this regard, high-frequency PHTS mutations targeting the Arg173 PTEN residue, which cause diminished protein stability, display increased ubiquitination at specific PTEN Lys

residues [46, 47]. In addition to the frequent impairment of PIP3 phosphatase activity in cells, changes in the PTEN cytoplasmic/nuclear ratio stands as one of the more common alterations in

PHTS patients. In our analysis, the P95R, Y177N, and T277A variants showed diminished nuclear accumulation in the background of PTEN 1-375, a recombinant proteoform that mimics one of the

PTEN C-terminal caspase-3 protein products [11]. When comparing PTEN (major cytoplasmic localization) with PTEN 1-375 (major nuclear localization; ref. [31]), the variants P95R, Y177N, and

T277A displayed enhanced cytoplasmic localization. We speculate that different regulatory mechanisms of PTEN subcellular localization could be affected by distinct PTEN PHTS mutations. We

did not detect significant alterations in the variants Q261E and D310G targeting the PTEN C2-domain, suggesting the existence of uncovered regulatory mechanisms of PTEN function. The Q261E

mutation incorporates a negative charge in the CBR3 loop at PTEN C2 domain, which has been involved in binding to membranes [14]. This makes possible altered membrane binding properties for

the PTEN Q261E variant. On the other hand, the D310G mutation removes a negative charge near the unstructured loop at the C2 domain, whose functions are unknown. The implementation of

alternative PTEN function experimental assays will be central to unveil the potential pathogenicity of these types of mutations. In this regard, the T26I and P96R variants displayed

diminished caspase-3 cleavage at Asp301 at the C2-domain unstructured loop, as monitored using a specific anti-PTEN Asp301-cleaved antibody. This could be a consequence of impaired catalytic

activity [11], but it could also be the result of subtle alterations in protein-protein interactions and subcellular localization. The regulation and functional consequences of Asp301 PTEN

cleavage are unknown, which deserves dedicated studies. In this regard, the PTEN Asp301 caspase-3 cleavage is not regulated by PTEN C-terminal tail phosphorylation by CK2, distinct to the

PTEN caspase-3 cleavage at the PTEN C-tail [11]. PTEN structure and intrinsic phosphatase activity is maintained in the absence of the C2-domain unstructured loop (residues 286-309)

containing the Asp301 residue [14], suggesting a non-essential role for this region in PTEN enzymatic activity. However, amino acid substitution variants targeting PTEN residues 286-309 are

found in PHTS patients and in tumors, suggesting an active regulatory role for the C2-domain unstructured loop linked to pathogenesis. In this regard, this loop contains the Lys289 residue,

which is targeted by mutation in PHTS and whose ubiquitination is important for facilitating PTEN nuclear import [48], an event that has been related with chemoresistance to temozolomide in

glioblastoma cells [49]. Also of interest, PTEN caspase cleavage has been associated with resistance to cisplatin treatment in the A2780 ovarian cancer cell line [13]. Further work is

necessary to elucidate the potential role of alterations in PTEN caspase-3 cleavage or PTEN nuclear import in PHTS pathogenesis and resistance to anti-cancer chemotherapy. DATA AVAILABILITY

The datasets generated during and/or analysed during the current study are available in the LOVD gene variant database (http://www.lovd.nl/3.0/home), with the following accession ID:

0000352584 (T26I), 0000878862 (P95R), 0000878883 (Y177N), 0000878885 (Q261E), 0000878886 (T277A), 0000878890 (D310G). REFERENCES * Yehia L, Keel E, Eng C. The Clinical Spectrum of PTEN

Mutations. Annu Rev Med. 2020;71:103–16. Article  CAS  PubMed  Google Scholar  * Ngeow J, Eng C. PTEN in Hereditary and Sporadic Cancer. Cold Spring Harbor Perspect Med. 2020;1:a036087. *

Pulido R, Mingo J, Gaafar A, Nunes-Xavier CE, Luna S, Torices L, et al. Precise Immunodetection of PTEN Protein in Human Neoplasia. Cold Spring Harb Perspect Med. 2019;9:a036293. Article 

CAS  PubMed  PubMed Central  Google Scholar  * Lee YR, Chen M, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nat Rev Mol cell Biol.

2018;19:547–62. Article  CAS  PubMed  Google Scholar  * Pulido R. PTEN: a yin-yang master regulator protein in health and disease. Methods. 2015;77-78:3–10. Article  CAS  PubMed  Google

Scholar  * A Papa, PP Pandolfi. Phosphatase-independent functions of the tumor suppressor PTEN. In: Neel B, Tonks N, editors. Protein Tyrosine Phosphatases in Cancer. New York, NY: Springer;

2016;247–60. * Bononi A, Pinton P. Study of PTEN subcellular localization. Methods. 2015;77-78:92–103. Article  CAS  PubMed  PubMed Central  Google Scholar  * Misra S, Ghosh G, Chowdhury

SG, Karmakar P. Non-canonical function of nuclear PTEN and its implication on tumorigenesis. DNA Repair (Amst). 2021;107:103197. Article  CAS  PubMed  Google Scholar  * Leslie NR, Kriplani

N, Hermida MA, Alvarez-Garcia V, Wise HM. The PTEN protein: cellular localization and post-translational regulation. Biochemical Soc Trans. 2016;44:273–8. Article  CAS  Google Scholar  *

Sellars E, Gabra M, Salmena L. The Complex Landscape of PTEN mRNA Regulation. Cold Spring Harb Perspect Med. 2020;10:a036236. Article  CAS  PubMed  PubMed Central  Google Scholar  * Torres

J, Rodriguez J, Myers MP, Valiente M, Graves JD, Tonks NK, et al. Phosphorylation-regulated cleavage of the tumor suppressor PTEN by caspase-3: implications for the control of protein

stability and PTEN-protein interactions. J Biol Chem. 2003;278:30652–60. Article  CAS  PubMed  Google Scholar  * Gil A, Andrés-Pons A, Pulido R. Nuclear PTEN: a tale of many tails. Cell

death Differ. 2007;14:395–9. Article  CAS  PubMed  Google Scholar  * Singh M, Chaudhry P, Fabi F, Asselin E. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer

cells: a potential mechanism of chemoresistance. BMC Cancer. 2013;13:233. Article  CAS  PubMed  PubMed Central  Google Scholar  * Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T,

Shi Y, et al. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell. 1999;99:323–34. Article  CAS  PubMed

  Google Scholar  * Fragoso R, Barata JT. Kinases, tails and more: Regulation of PTEN function by phosphorylation. Methods. 2014. * Sotelo NS, Schepens JT, Valiente M, Hendriks WJ, Pulido R.

PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13. Methods. 2015;77-78:147–56. Article  CAS  PubMed  Google Scholar  * Wang K, Liu J, Li YL, Li JP, Zhang R.

Ubiquitination/de-ubiquitination: A promising therapeutic target for PTEN reactivation in cancer. Biochim Biophys Acta Rev Cancer. 2022;1877:188723. Article  CAS  PubMed  Google Scholar  *

Matreyek KA, Starita LM, Stephany JJ, Martin B, Chiasson MA, Gray VE, et al. Multiplex assessment of protein variant abundance by massively parallel sequencing. Nat Genet. 2018;50:874–82.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Mighell TL, Evans-Dutson S, O’Roak BJ. A Saturation Mutagenesis Approach to Understanding PTEN Lipid Phosphatase Activity and

Genotype-Phenotype Relationships. Am J Hum Genet. 2018;102:943–55. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mingo J, Rodriguez-Escudero I, Luna S, Fernandez-Acero T, Amo L,

Jonasson AR, et al. A pathogenic role for germline PTEN variants which accumulate into the nucleus. Eur J Hum Genet: EJHG. 2018;26:1180–7. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Rodríguez-Escudero I, Oliver MD, Andrés-Pons A, Molina M, Cid VJ, Pulido R. A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related

syndromes. Hum Mol Genet. 2011;20:4132–42. Article  PubMed  Google Scholar  * Spinelli L, Black FM, Berg JN, Eickholt BJ, Leslie NR. Functionally distinct groups of inherited PTEN mutations

in autism and tumour syndromes. J Med Genet. 2015;52:128–34. Article  CAS  PubMed  Google Scholar  * Post KL, Belmadani M, Ganguly P, Meili F, Dingwall R, McDiarmid TA, et al. Multi-model

functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction. Nat Commun. 2020;11:2073. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Chao JT, Hollman R, Meyers WM, Meili F, Matreyek KA, Dean P, et al. A Premalignant Cell-Based Model for Functionalization and Classification of PTEN

Variants. Cancer Res. 2020;80:2775–89. Article  CAS  PubMed  Google Scholar  * Ganguly P, Madonsela L, Chao JT, Loewen CJR, O’Connor TP, Verheyen EM, et al. A scalable Drosophila assay for

clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation. PLoS Genet. 2021;17:e1009774. Article  CAS  PubMed  PubMed Central  Google Scholar 

* Smith IN, Thacker S, Seyfi M, Cheng F, Eng C. Conformational Dynamics and Allosteric Regulation Landscapes of Germline PTEN Mutations Associated with Autism Compared to Those Associated

with Cancer. Am J Hum Genet. 2019;104:861–78. Article  CAS  PubMed  PubMed Central  Google Scholar  * Cid VJ, Rodríguez-Escudero I, Andrés-Pons A, Romá-Mateo C, Gil A, den Hertog J, et al.

Assessment of PTEN tumor suppressor activity in nonmammalian models: the year of the yeast. Oncogene. 2008;27:5431–42. Article  CAS  PubMed  Google Scholar  * Leslie NR, Longy M. Inherited

PTEN mutations and the prediction of phenotype. Semin Cell Developmental Biol. 2016;52:30–8. Article  CAS  Google Scholar  * Pena-Couso L, Ercibengoa M, Mercadillo F, Gomez-Sanchez D,

Inglada-Perez L, Santos M, et al. Considerations on diagnosis and surveillance measures of PTEN hamartoma tumor syndrome: clinical and genetic study in a series of Spanish patients. Orphanet

J Rare Dis. 2022;17:85. Article  PubMed  PubMed Central  Google Scholar  * Andrés-Pons A, Rodríguez-Escudero I, Gil A, Blanco A, Vega A, Molina M, et al. In vivo functional analysis of the

counterbalance of hyperactive phosphatidylinositol 3-kinase p110 catalytic oncoproteins by the tumor suppressor PTEN. Cancer Res. 2007;67:9731–9. Article  PubMed  Google Scholar  * Gil A,

Andrés-Pons A, Fernández E, Valiente M, Torres J, Cervera J, et al. Nuclear localization of PTEN by a Ran-dependent mechanism enhances apoptosis: Involvement of an N-terminal nuclear

localization domain and multiple nuclear exclusion motifs. Mol Biol Cell. 2006;17:4002–13. Article  CAS  PubMed  PubMed Central  Google Scholar  * Andrés-Pons A, Gil A, Oliver MD, Sotelo NS,

Pulido R. Cytoplasmic p27Kip1 counteracts the pro-apoptotic function of the open conformation of PTEN by retention and destabilization of PTEN outside of the nucleus. Cell Signal.

2012;24:577–87. Article  PubMed  Google Scholar  * Mingo J, Erramuzpe A, Luna S, Aurtenetxe O, Amo L, Diez I, et al. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative

Approach to Generate Amino Acid Substitution Collections. PloS One. 2016;11:e0160972. Article  PubMed  PubMed Central  Google Scholar  * Rodriguez-Escudero I, Fernandez-Acero T, Bravo I,

Leslie NR, Pulido R, Molina M, et al. Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo. Methods. 2015;77-78:172–9. Article  CAS  PubMed  Google Scholar  *

Rodríguez-Escudero I, Roelants FM, Thorner J, Nombela C, Molina M, Cid VJ. Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast. Biochemical J. 2005;390:613–23. Pt 2 Article 

Google Scholar  * Nunes-Xavier CE, Pulido R. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome. Methods Mol Biol. 2016;1447:25–37. Article  CAS  PubMed  Google

Scholar  * Andrés-Pons A, Valiente M, Torres J, Gil A, Roglá I, Ripoll F, et al. Functional definition of relevant epitopes on the tumor suppressor PTEN protein. Cancer Lett.

2005;223:303–12. Article  PubMed  Google Scholar  * Sotelo NS, Valiente M, Gil A, Pulido R. A functional network of the tumor suppressors APC, hDlg, and PTEN, that relies on recognition of

specific PDZ-domains. J Cell Biochem. 2012;113:2661–70. Article  CAS  PubMed  Google Scholar  * Tolkacheva T, Boddapati M, Sanfiz A, Tsuchida K, Kimmelman AC, Chan AM. Regulation of PTEN

binding to MAGI-2 by two putative phosphorylation sites at threonine 382 and 383. Cancer Res. 2001;61:4985–9. CAS  PubMed  Google Scholar  * Torres J, Pulido R. The tumor suppressor PTEN is

phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. The. J Biol Chem. 2001;276:993–8. Article  CAS  PubMed  Google

Scholar  * Yang JM, Schiapparelli P, Nguyen HN, Igarashi A, Zhang Q, Abbadi S, et al. Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes

protein stability and nuclear localization. Oncogene. 2017;36:3673–85. Article  CAS  PubMed  PubMed Central  Google Scholar  * Ho J, Cruise ES, Dowling RJO, Stambolic V. PTEN Nuclear

Functions. Cold Spring Harb Perspect Med. 2020;10:a036079. Article  CAS  PubMed  PubMed Central  Google Scholar  * Gil A, Rodriguez-Escudero I, Stumpf M, Molina M, Cid VJ, Pulido R. A

functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity. PloS one. 2015;10:e0119287. Article  PubMed  PubMed Central  Google

Scholar  * Wong CW, Wang Y, Liu T, Li L, Cheung SKK, Or PM, et al. Autism-associated PTEN missense mutation leads to enhanced nuclear localization and neurite outgrowth in an induced

pluripotent stem cell line. The. FEBS J. 2020;287:4848–61. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang H, Karikomi M, Naidu S, Rajmohan R, Caserta E, Chen HZ, et al.

Allele-specific tumor spectrum in pten knockin mice. Proc Natl Acad Sci USA. 2010;107:5142–7. Article  CAS  PubMed  PubMed Central  Google Scholar  * Guo Y, He J, Zhang H, Chen R, Li L, Liu

X, et al. Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression. Oncogene. 2022;41:4877–92. Article  CAS  PubMed  Google Scholar  * Pearce W, Kessaris N,

Leslie NR, Vanhaesebroeck B, Tibarewal P, Classen G, et al. Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo

approaches. Mol cancer Res: MCR. 2020;18:B22. Article  Google Scholar  * Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, Yang H, et al. Ubiquitination regulates PTEN nuclear

import and tumor suppression. Cell 2007;128:141–56. Article  CAS  PubMed  PubMed Central  Google Scholar  * Dong L, Li Y, Liu L, Meng X, Li S, Han D, et al. Smurf1 Suppression Enhances

Temozolomide Chemosensitivity in Glioblastoma by Facilitating PTEN Nuclear Translocation. Cells. 2022;11:3302. Article  CAS  PubMed  PubMed Central  Google Scholar  * Busa T, Milh M,

Degardin N, Girard N, Sigaudy S, Longy M, et al. Clinical presentation of PTEN mutations in childhood in the absence of family history of Cowden syndrome. Eur J Paediatr Neurol: EJPN: Off J

Eur Paediatr Neurol Soc. 2015;19:188–92. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Gustavo Pérez‐Nanclares and Ana Belén de la Hoz (Genetics-Genomics Core

facility, Biocruces Bizkaia Health Research Institute) for their expert assistance with DNA sequencing., and Javier Díez Garcı́a (Microscopy core facility, Biocruces Bizkaia Health Research

Institute) for expert microscopy technical support. FUNDING This work has been supported in part by grant BBH-19-001 (to RP) from PTEN Research Foundation (United Kingdom); grants

SAF2016-79847-R (to RP and JIL), and PID2019-105342GB-I00 (to VJC and MM) from Ministerio de Economía y Competitividad (Spain and The European Regional Development Fund); and grant

S2017/BMD‐3691(InGEMICS‐CM) from Comunidad de Madrid and European Structural and Investment Funds (to VJC and MM). LT has been the recipient of a predoctoral fellowship from Asociación

Española Contra el Cáncer (AECC, Junta Provincial de Bizkaia, Spain). JM has been the recipient of a predoctoral fellowship (PRE_2014_1_285) from Gobierno Vasco, Departamento de Educación

(Basque Country, Spain). CN-X is the recipient of a Miguel Servet Research Contract from Instituto de Salud Carlos III (grant number CP20/00008). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS

* Biocruces Bizkaia Health Research Institute, Barakaldo, Spain Leire Torices, Janire Mingo, Sandra Luna, Caroline E. Nunes-Xavier, José I. López & Rafael Pulido * Departamento de

Microbiología y Parasitología, Facultad de Farmacia, UCM & Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain Isabel Rodríguez-Escudero, Teresa

Fernández-Acero, María Molina & Víctor J. Cid * Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Caroline E. Nunes-Xavier * Department of Pathology, Cruces

University Hospital, Barakaldo, Spain José I. López * Familial Cancer Clinical Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Fátima Mercadillo, María Currás & 

Miguel Urioste * Ikerbasque, The Basque Foundation for Science, Bilbao, Spain Rafael Pulido Authors * Leire Torices View author publications You can also search for this author inPubMed 

Google Scholar * Janire Mingo View author publications You can also search for this author inPubMed Google Scholar * Isabel Rodríguez-Escudero View author publications You can also search

for this author inPubMed Google Scholar * Teresa Fernández-Acero View author publications You can also search for this author inPubMed Google Scholar * Sandra Luna View author publications

You can also search for this author inPubMed Google Scholar * Caroline E. Nunes-Xavier View author publications You can also search for this author inPubMed Google Scholar * José I. López

View author publications You can also search for this author inPubMed Google Scholar * Fátima Mercadillo View author publications You can also search for this author inPubMed Google Scholar

* María Currás View author publications You can also search for this author inPubMed Google Scholar * Miguel Urioste View author publications You can also search for this author inPubMed 

Google Scholar * María Molina View author publications You can also search for this author inPubMed Google Scholar * Víctor J. Cid View author publications You can also search for this

author inPubMed Google Scholar * Rafael Pulido View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS LT, JM, IR-E, TF-A, and SL designed and

performed experiments, and performed data analysis. CEN-X designed experiments, supervised the work and performed data analysis. JIL and MM supervised the work. FM, MC, and MU shared

information and provided feedback. VJC and RP designed experiments, performed data analysis and wrote the manuscript. All authors revised the manuscript. CORRESPONDING AUTHOR Correspondence

to Rafael Pulido. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to

this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the

terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Torices, L., Mingo, J., Rodríguez-Escudero, I. _et al._ Functional

analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease. _Eur J Hum Genet_ 31, 568–577 (2023).

https://doi.org/10.1038/s41431-022-01265-w Download citation * Received: 16 June 2022 * Revised: 01 December 2022 * Accepted: 06 December 2022 * Published: 21 December 2022 * Issue Date: May

2023 * DOI: https://doi.org/10.1038/s41431-022-01265-w SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable

link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative