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doi: 10.1242/10.1242/jcs.00448

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Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4,5-biphosphate

Guillaume Bompard{ddagger},1,*, Marianne Martin2, Christian Roy2, Françoise Vignon1 and Gilles Freiss1

1 Inserm U540, Endocrinologie Moléculaire et Cellulaire des Cancers, Montpellier, France
2 Dynamique Molèculaire des Interactions Membranaires, Université Montpellier II, Unité Mixte de Recherche (UMR) CNRS 5539, Montpellier Cedex 5, France.



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  		Fig. 1. PTPL1 expression constructs. (A) Schematic representation of the PTPL1 expression constructs used. The modular protein domains are indicated in square. Protein segments produced by transient expression are indicated within parentheses. Numbers correspond to the first and last amino acids positions according to the PTPL1 sequence (EMBL accession X80289). (B) Expression of PTPL1-FERM constructs. COS1 cells were transiently transfected with HA-epitope tagged PTPL1-FERM constructs: wt (lane1), KN1 (lane 2), KN2 (lane 3) or KN1-2 (lane 4) mutants. Cells were lysed in SDS sample buffer, fractionated on a 12.5% SDS-PAGE and expression was revealed after western blotting with anti-HA antibody. (C) Expression of full-length PTPL1 constructions. COS1 cells were transiently transfected with HA-epitope tagged PTPL1 constructs: wt (lane1), KN1-2 (lane 2) or {Delta}FERM (lane 3) mutants. Expression was revealed as described above after fractionation on a 7% SDS-PAGE gel. Molecular weight markers (left) are experssed in kDa. *Nonspecific band revealed with anti-HA antibody.

 


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  		Fig. 2. The FERM domain of PTPL1 is necessary and sufficient for its apical localization and microvilli association in HeLa cells. (A-C) Full-length PTPL1, (D-F) PTPL1-FERM domain, and (G-I) PTPL1 {Delta}FERM with HA-epitope tags were transiently expressed in HeLa epithelial cells (see Fig. 1). Cells were treated for (left) indirect FITC localization of PTPL1 constructs with anti-HA antibody and (middle) F-actin with TRITC-coupled phalloidin. Images represent merges of horizontal confocal sections. Vertical xz sections at the level of the dotted lines are presented under each figure. Co-localizations of PTPL1 constructs and actin are represented by yellow. Full-length PTPL1 is (A) located at the apical face of cell membrane and (C) co-localized with F-actin at the level of dorsal microvilli (arrow) and lateral cell membranes. (F) The PTPL1-FERM domain displayed the same subcellular localization (arrow) as the full-length enzyme. (D) Cell extensions were also observed (arrowhead). On the contrary, (G) PTPL1 {Delta}FERM was found mainly in the cytosol and enriched around the nucleus (arrows). Bars, 20 µm.

 


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  		Fig. 3. Alignment of FERM domain sequences from PTPs and ezrin. The aligned FERM domains are human PTPH1 (hPTPH1), Caenorhabditis elegans PTP-FERM (PTP-FERM), human PTPL1 (hPTPL1) and human ezrin (hEzrin). Conserved amino acid residues from at least two sequences are highlighted in black when identical or in gray when encompassing to the same family. Bold bars represent localization of the two PtdIns(4,5)P2-binding motifs identified in ezrin. Subdomains A, B and C of ezrin are indicated by thin bars.

 


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  		Fig. 4. The localization of the PTPL1-FERM domain is altered by mutation of the two potential PtdIns(4,5)P2-binding sites in COS1 cells. COS1 cells were transfected with (A) HA-epitope tagged wild-type FERM domain, (B) KN1-FERM mutant (K645, 646, 647N), (C) KN2-FERM mutant (K824, 825, 830, 831, 832N) and (D) KN1-2-FERM mutant (combination of KN1 and KN2 mutations) and treated for immunolocalization of HA-epitope tagged constructs as described in Fig. 2. (A-C) Transfected wild-type FERM domain, KN1 and KN2 mutants were associated with the plasma membrane, dorsal microvilli and induced membrane extensions (arrowheads). (D) KN1-2 mutant of FERM domain was mainly localized in the cytoplasm (arrowhead). Bars, 10 µm.

 


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  		Fig. 5. Mutation of both potential PtdIns(4,5)P2-binding motifs altered subcellular localization of FERM domain as well as full-length PTPL1 in HeLa cells. HA-epitope tagged (A-C) PTPL1-FERM domain or (D-F) full-length PTPL1 carrying KN1-2 mutations were transiently expressed in HeLa cells. Cells were treated for indirect immunofluorescence of (A,D) PTPL1 constructs and (B,E) F-actin as described in Fig. 2. (C,F) Dual localizations are merged in color. Images represent merges of horizontal confocal planes. (A,E) Both constructs displayed a similar cytosolic localization enriched around nucleus (arrows). Bars, 20 µm.

 


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  		Fig. 6. PTPL1-FERM interaction with PIPs. 35S-labeled PTPL1-FERM domain (wt) and its KN1, KN2, KN1-2 mutants were produced by in vitro transcription-coupled translation system. (A) 4000 cpm of each labeled recombinant protein were analyzed on a 10% SDS/PAGE and submitted to autoradiography. (B) 40,000 cpm of each labeled protein were incubated with PIP-Strips (Echelon Biosciences) plotted with 100 pmol of PtdIns, PtdIns(3)P [PI(3)P], PtdIns(4)P [PI(4)P], PtdIns(5)P [PI(5)P], PtdIns(3,4)P2 [PI(3,4)P2], PtdIns(3,5)P2 [PI(3,5)P2], PtdIns(4,5)P2 [PtdIns(4,5)P2], PtdIns(3,4,5)P3 [PI(3,4,5)P3], phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), lysophosphatidic acid (LPA), lysophosphacholine (LPC), sphingosine-1-phosphate (S1P) and phosphatic acid (PA). After three washes, PIP-strips were submitted to autoradiography.

 


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  		Fig. 7. Subcellular distribution of transfected mutated or wild-type PTPL1-FERM domain or full-length PTPL1. Transfected COS1 cells were fractionated and the resulting fractions were analyzed on a 10% SDS/PAGE and immunoblotted. F-actin and intermediate filaments containing vimentin were recovered in the particulate fraction (P, pellet), whereas G-actin and depolymerised tubulin were recovered in the soluble fraction (S, supernatant). The equal amount of proteins loaded in each fraction was checked by Coomassie blue staining. (A) Transfection of FERM domains. Transfected HA-epitope tagged wild-type FERM domain was found to be strongly associated with the P fraction as detected after immunoblotting with anti-HA monoclonal antibody. KN1-FERM mutant displayed an equivalent distribution. KN2-and KN1-2-FERM mutants were only detected in the S fraction. (B) Transfection of full-length PTPL1. Transfected HA-epitope tagged PTPL1 was mainly recovered in the S fraction and significantly in the P fraction. KN1-2 or {Delta}FERM mutants were only detected in the S fraction. To ensure proper fractionation, membranes were immunoblotted with anti-actin polyclonal antibody, anti-vimentin and anti-tubulin monoclonal antibodies.

 


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  		Fig. 8. Effects of FERM mutations (KN1-2 and {Delta}FERM) and neomycin treatment on the association of PTPL1 with the cytoskeletal fraction. The amount of PTPL1 and its mutants with the particulate (P) fraction after COS1 cell fractionation (see Figs 7 and 9) treated or not with neomycin was quantified after densitometry of several immunoblots. White and black columns represent the percentage of PTPL1 associated with the cytoskeletal fraction in the absence or presence of neomycin treatment, respectively. Association of wild-type PTPL1 with the cytoskeletal fraction was significantly different after neomycin treatment (P<0.005, Student's t-test, n=10) and almost unchanged for KN1-2 and {Delta}FERM mutants (P=0.33 and P=0.42, respectively, Student's t-test, n=9). Mean±s.d. of n experiments (numbers in parentheses).

 


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  		Fig. 9. Effect of neomycin treatment on subcellular distribution of PTPL1. (A) Transfection of ezrin-FERM domain. COS1 cells were transfected with VSV-G-epitope tagged N-terminal part of ezrin (FERM domain, 1-310 aa) and were fractionated after treatment with or without 10 mM neomycin for 24 hours. Association of ezrin-FERM domain with particulate fraction was strongly inhibited by such cell treatment. (B) 35S-labeled PTPL1-FERM domain was incubated in TBST 3% BSA with PIP-Strips (Echelon Biosciences) pre-saturated with TBST 3% BSA [C] or TBST 3% BSA containing 1 mM neomycin [Neo]. (C) Transfection of PTPL1-FERM domains. COS1 cells were transfected with either HA-epitope tagged wild-type FERM domain or KN1-2-FERM domain, treated or not with neomycin as described above. Similarly to ezrin-FERM domain, association of wild-type FERM domain with P fraction was impaired by neomycin treatment. On the contrary, the distribution of the KN1-2-FERM mutant was not affected by such a treatment. (D) Transfection of full-length PTPL1. Similarly, COS1 cells were transfected with HA-epitope tagged wild-type PTPL1, PTPL1 KN1-2 or PTPL1 {Delta}FERM, and fractionated after neomycin treatment. Neomycin treatment strongly inhibited the association of wild-type PTPL1 with the P fraction. Moreover, KN1-2 and {Delta}FERM mutant distributions were not affected.

 

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