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Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Aicher B, Lerch MM, Müller T, Schilling J, Ullrich A - J. Cell Biol. (1997)

Bottom Line: Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro.Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain.We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.

ABSTRACT
Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

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Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. (A) Schematic  representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAREC and 322 recognize the E subunits of LAR and PTPσ, respectively. (B) LAR or PTPσ  were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10−5 M A23187, as indicated. Control  cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE (TRITON) or after binding  to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera  specific for the COOH terminus of LAR and PTPσ (320), the NH2 terminus of PTPσ (322), or the COOH terminus of the LAR E subunit (αLAREC). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and  PTPσ subunits. (C) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [35S]methionine (16 h) and incubated  with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ (320) or with  nonimmune serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h.  Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.
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Figure 1: Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. (A) Schematic representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAREC and 322 recognize the E subunits of LAR and PTPσ, respectively. (B) LAR or PTPσ were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10−5 M A23187, as indicated. Control cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE (TRITON) or after binding to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera specific for the COOH terminus of LAR and PTPσ (320), the NH2 terminus of PTPσ (322), or the COOH terminus of the LAR E subunit (αLAREC). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and PTPσ subunits. (C) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [35S]methionine (16 h) and incubated with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ (320) or with nonimmune serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h. Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.

Mentions: LAR and PTPσ are highly related PTPases whose rat homologues display a sequence identity of 79% in their proximal membrane PTPase domain, 90% in their COOH-terminal PTPase domain, and 57% in their extracellular domain (Zhang et al., 1994). LAR contains three Ig- and eight FNIII-like domains in the extracellular domain and two intracellular PTPase domains. Three splice variants of PTPσ are known so far. The rat protein we analyzed (Pan et al., 1993; Walton et al., 1993; Yan et al., 1993) differs from LAR in so far as it lacks the FNIII-like domains four through seven. While it was shown that LAR was expressed in two subunits (Streuli et al., 1992; Yu et al., 1992), the biosynthesis of PTPσ has not yet been studied. We hypothesized that PTPσ would be processed in a manner that is analogous or similar to the processing of LAR because a polyclonal antiserum directed against one of its FNIII-like domains recognized a protein of ∼100 kD instead of the 168 kD that would have been predicted from the full length sequence of PTPσ (Yan et al., 1993; Rotin et al., 1994). Fig. 1 A shows the schematic structure of LAR and PTPσ, the proposed biosynthesis of PTPσ, and the recognition sites of subunit-specific antibodies used in this study.


Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Aicher B, Lerch MM, Müller T, Schilling J, Ullrich A - J. Cell Biol. (1997)

Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. (A) Schematic  representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAREC and 322 recognize the E subunits of LAR and PTPσ, respectively. (B) LAR or PTPσ  were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10−5 M A23187, as indicated. Control  cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE (TRITON) or after binding  to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera  specific for the COOH terminus of LAR and PTPσ (320), the NH2 terminus of PTPσ (322), or the COOH terminus of the LAR E subunit (αLAREC). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and  PTPσ subunits. (C) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [35S]methionine (16 h) and incubated  with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ (320) or with  nonimmune serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h.  Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.
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Figure 1: Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. (A) Schematic representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAREC and 322 recognize the E subunits of LAR and PTPσ, respectively. (B) LAR or PTPσ were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10−5 M A23187, as indicated. Control cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE (TRITON) or after binding to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera specific for the COOH terminus of LAR and PTPσ (320), the NH2 terminus of PTPσ (322), or the COOH terminus of the LAR E subunit (αLAREC). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and PTPσ subunits. (C) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [35S]methionine (16 h) and incubated with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ (320) or with nonimmune serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h. Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.
Mentions: LAR and PTPσ are highly related PTPases whose rat homologues display a sequence identity of 79% in their proximal membrane PTPase domain, 90% in their COOH-terminal PTPase domain, and 57% in their extracellular domain (Zhang et al., 1994). LAR contains three Ig- and eight FNIII-like domains in the extracellular domain and two intracellular PTPase domains. Three splice variants of PTPσ are known so far. The rat protein we analyzed (Pan et al., 1993; Walton et al., 1993; Yan et al., 1993) differs from LAR in so far as it lacks the FNIII-like domains four through seven. While it was shown that LAR was expressed in two subunits (Streuli et al., 1992; Yu et al., 1992), the biosynthesis of PTPσ has not yet been studied. We hypothesized that PTPσ would be processed in a manner that is analogous or similar to the processing of LAR because a polyclonal antiserum directed against one of its FNIII-like domains recognized a protein of ∼100 kD instead of the 168 kD that would have been predicted from the full length sequence of PTPσ (Yan et al., 1993; Rotin et al., 1994). Fig. 1 A shows the schematic structure of LAR and PTPσ, the proposed biosynthesis of PTPσ, and the recognition sites of subunit-specific antibodies used in this study.

Bottom Line: Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro.Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain.We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.

ABSTRACT
Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

Show MeSH
Related in: MedlinePlus