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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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Colocalization of  LAR and PTPσ with plakoglobin after ionophore and TPA  treatment. A431 cells were  fixed and labeled for plakoglobin (A, C, E, and G) or double  labeled with anti-plakoglobin  and antiserum 320 (COOH  terminus of LAR and PTPσ;  B, D, F, and H) as described in  Materials and Methods. Laser  confocal fluorescence images  were superimposed on a transmission image to show protein  localization together with cellular and junctional morphology. Red pseudocolor indicates  monoclonal anti-plakoglobin  antibody; green, polyclonal  320 antiserum; and yellow, regions of colocalization of both.  Prefixation treatment was as  follows: (A and B) controls; (C  and D) A23187 (10−5 M) for  30 min; (E and F) TPA (1 μM)  for 40 min; (G and H) TPA (1  μM) for 240 min. Note rapid  internalization of label and  disruption of cell–cell contacts  in C and D. While 40 min of  TPA neither disrupted intercellular junctions nor altered  plakoglobin localization (E),  antibody 320 label was already  found in the cytosol (F). After  4 h of TPA treatment, significant plakoglobin label (G) and  even more antibody 320 label  (H) were found internalized in  comparison to the label remaining at the plasma membrane or  at cell–cell adhesions. Bar, 25 μm.
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Figure 9: Colocalization of LAR and PTPσ with plakoglobin after ionophore and TPA treatment. A431 cells were fixed and labeled for plakoglobin (A, C, E, and G) or double labeled with anti-plakoglobin and antiserum 320 (COOH terminus of LAR and PTPσ; B, D, F, and H) as described in Materials and Methods. Laser confocal fluorescence images were superimposed on a transmission image to show protein localization together with cellular and junctional morphology. Red pseudocolor indicates monoclonal anti-plakoglobin antibody; green, polyclonal 320 antiserum; and yellow, regions of colocalization of both. Prefixation treatment was as follows: (A and B) controls; (C and D) A23187 (10−5 M) for 30 min; (E and F) TPA (1 μM) for 40 min; (G and H) TPA (1 μM) for 240 min. Note rapid internalization of label and disruption of cell–cell contacts in C and D. While 40 min of TPA neither disrupted intercellular junctions nor altered plakoglobin localization (E), antibody 320 label was already found in the cytosol (F). After 4 h of TPA treatment, significant plakoglobin label (G) and even more antibody 320 label (H) were found internalized in comparison to the label remaining at the plasma membrane or at cell–cell adhesions. Bar, 25 μm.

Mentions: We used antibodies directed against plakoglobin (γ-catenin), a protein localized at the intracellular site of adherens junctions and desmosomes (Cowin et al., 1986), to study whether LAR and PTPσ colocalize to these specialized areas and to investigate whether the internalization of the phosphatases from the plasma membrane is paralleled by a dissociation of plakoglobin from this site. Plakoglobin (Fig. 9 A) was detected along cell–cell contacts of neighboring A431 cells, strongly colocalized with antiserum 320 label (Fig. 9 B), and therefore identified the subcellular site of LAR and PTPσ as adherens junctions and desmosomes. Upon ionophore treatment, plakoglobin (Fig. 9 C) and the phosphatase domains were internalized in parallel from the rapidly dissociating intercellular junctions (Fig. 9 D). Incubation with TPA for 40 min left the localization of anti-plakoglobin labeling unaffected (Fig. 9 E) but induced a significant yet incomplete internalization of 320 label (Fig. 9 F). Extended time course experiments with TPA treatment up to 4 h showed that plakoglobin, in agreement with studies by Fabre and DeHerreros (1993), is redistributed from the plasma membrane to the cytosol (Fig. 9 G), although at a much slower rate and not in parallel with the intracellular phosphatase domain (Fig. 9 H). When antibodies directed against the extracellular domains of LAR and PTPσ were used for immunolabeling, both ionophore treatment and TPA incubation induced a complete disappearance of fluorescence label (data not shown).


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)

Colocalization of  LAR and PTPσ with plakoglobin after ionophore and TPA  treatment. A431 cells were  fixed and labeled for plakoglobin (A, C, E, and G) or double  labeled with anti-plakoglobin  and antiserum 320 (COOH  terminus of LAR and PTPσ;  B, D, F, and H) as described in  Materials and Methods. Laser  confocal fluorescence images  were superimposed on a transmission image to show protein  localization together with cellular and junctional morphology. Red pseudocolor indicates  monoclonal anti-plakoglobin  antibody; green, polyclonal  320 antiserum; and yellow, regions of colocalization of both.  Prefixation treatment was as  follows: (A and B) controls; (C  and D) A23187 (10−5 M) for  30 min; (E and F) TPA (1 μM)  for 40 min; (G and H) TPA (1  μM) for 240 min. Note rapid  internalization of label and  disruption of cell–cell contacts  in C and D. While 40 min of  TPA neither disrupted intercellular junctions nor altered  plakoglobin localization (E),  antibody 320 label was already  found in the cytosol (F). After  4 h of TPA treatment, significant plakoglobin label (G) and  even more antibody 320 label  (H) were found internalized in  comparison to the label remaining at the plasma membrane or  at cell–cell adhesions. Bar, 25 μm.
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Figure 9: Colocalization of LAR and PTPσ with plakoglobin after ionophore and TPA treatment. A431 cells were fixed and labeled for plakoglobin (A, C, E, and G) or double labeled with anti-plakoglobin and antiserum 320 (COOH terminus of LAR and PTPσ; B, D, F, and H) as described in Materials and Methods. Laser confocal fluorescence images were superimposed on a transmission image to show protein localization together with cellular and junctional morphology. Red pseudocolor indicates monoclonal anti-plakoglobin antibody; green, polyclonal 320 antiserum; and yellow, regions of colocalization of both. Prefixation treatment was as follows: (A and B) controls; (C and D) A23187 (10−5 M) for 30 min; (E and F) TPA (1 μM) for 40 min; (G and H) TPA (1 μM) for 240 min. Note rapid internalization of label and disruption of cell–cell contacts in C and D. While 40 min of TPA neither disrupted intercellular junctions nor altered plakoglobin localization (E), antibody 320 label was already found in the cytosol (F). After 4 h of TPA treatment, significant plakoglobin label (G) and even more antibody 320 label (H) were found internalized in comparison to the label remaining at the plasma membrane or at cell–cell adhesions. Bar, 25 μm.
Mentions: We used antibodies directed against plakoglobin (γ-catenin), a protein localized at the intracellular site of adherens junctions and desmosomes (Cowin et al., 1986), to study whether LAR and PTPσ colocalize to these specialized areas and to investigate whether the internalization of the phosphatases from the plasma membrane is paralleled by a dissociation of plakoglobin from this site. Plakoglobin (Fig. 9 A) was detected along cell–cell contacts of neighboring A431 cells, strongly colocalized with antiserum 320 label (Fig. 9 B), and therefore identified the subcellular site of LAR and PTPσ as adherens junctions and desmosomes. Upon ionophore treatment, plakoglobin (Fig. 9 C) and the phosphatase domains were internalized in parallel from the rapidly dissociating intercellular junctions (Fig. 9 D). Incubation with TPA for 40 min left the localization of anti-plakoglobin labeling unaffected (Fig. 9 E) but induced a significant yet incomplete internalization of 320 label (Fig. 9 F). Extended time course experiments with TPA treatment up to 4 h showed that plakoglobin, in agreement with studies by Fabre and DeHerreros (1993), is redistributed from the plasma membrane to the cytosol (Fig. 9 G), although at a much slower rate and not in parallel with the intracellular phosphatase domain (Fig. 9 H). When antibodies directed against the extracellular domains of LAR and PTPσ were used for immunolabeling, both ionophore treatment and TPA incubation induced a complete disappearance of fluorescence label (data not shown).

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