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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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Surface morphology of intercellular adhesions. A431 cells were fixed  and prepared for scanning  electron microscopy as described in Materials and  Methods. Prefixation treatment was as follows: (A) control; (B) vehicle (DMSO);  (C) EGTA (5 mM) for 45  min; (D) EGTA (5 mM) for  45 min plus A23187 (10−5 M)  for the last 30 min; (E)  A23187 (10−5 M) for 30 min;  (F) TPA (1 μM) for 40 min.  Note intact cell–cell contacts  in A, B, and F and largely disrupted intercellular junctions  in C, D, and E. Bar, 10 μm.
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Figure 8: Surface morphology of intercellular adhesions. A431 cells were fixed and prepared for scanning electron microscopy as described in Materials and Methods. Prefixation treatment was as follows: (A) control; (B) vehicle (DMSO); (C) EGTA (5 mM) for 45 min; (D) EGTA (5 mM) for 45 min plus A23187 (10−5 M) for the last 30 min; (E) A23187 (10−5 M) for 30 min; (F) TPA (1 μM) for 40 min. Note intact cell–cell contacts in A, B, and F and largely disrupted intercellular junctions in C, D, and E. Bar, 10 μm.

Mentions: The localization of LAR and PTPσ at cell–cell contacts and their partial or complete internalization raised the question whether and how EGTA, ionophore, and TPA had affected the structural integrity of these specialized membrane regions. On scanning electron microscopy, untreated and vehicle (DMSO)-treated A431 cells were found to have formed multiple adhesion complexes between neighboring cells (Fig. 8, A and B). Incubation with EGTA (Fig. 8 C), as well as subsequent treatment with ionophore (Fig. 8 D) or ionophore alone (Fig. 8 E) induced an almost complete disruption of intercellular junctions, the formation of multiple surface protrusions, and a rounded elevation of the normally flat cell body from the surface. 40 min of treatment with TPA, which was also associated with internalization of LAR and PTPσ on immunolabeling studies (Fig. 7 H), left the junctional morphology completely intact (Fig. 8 F). These observations indicated that only the EGTA- and ionophore-induced internalization was paralleled by a structural disruption of intercellular adhesions, whereas TPA-induced internalization occurred independently of this effect.


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)

Surface morphology of intercellular adhesions. A431 cells were fixed  and prepared for scanning  electron microscopy as described in Materials and  Methods. Prefixation treatment was as follows: (A) control; (B) vehicle (DMSO);  (C) EGTA (5 mM) for 45  min; (D) EGTA (5 mM) for  45 min plus A23187 (10−5 M)  for the last 30 min; (E)  A23187 (10−5 M) for 30 min;  (F) TPA (1 μM) for 40 min.  Note intact cell–cell contacts  in A, B, and F and largely disrupted intercellular junctions  in C, D, and E. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141638&req=5

Figure 8: Surface morphology of intercellular adhesions. A431 cells were fixed and prepared for scanning electron microscopy as described in Materials and Methods. Prefixation treatment was as follows: (A) control; (B) vehicle (DMSO); (C) EGTA (5 mM) for 45 min; (D) EGTA (5 mM) for 45 min plus A23187 (10−5 M) for the last 30 min; (E) A23187 (10−5 M) for 30 min; (F) TPA (1 μM) for 40 min. Note intact cell–cell contacts in A, B, and F and largely disrupted intercellular junctions in C, D, and E. Bar, 10 μm.
Mentions: The localization of LAR and PTPσ at cell–cell contacts and their partial or complete internalization raised the question whether and how EGTA, ionophore, and TPA had affected the structural integrity of these specialized membrane regions. On scanning electron microscopy, untreated and vehicle (DMSO)-treated A431 cells were found to have formed multiple adhesion complexes between neighboring cells (Fig. 8, A and B). Incubation with EGTA (Fig. 8 C), as well as subsequent treatment with ionophore (Fig. 8 D) or ionophore alone (Fig. 8 E) induced an almost complete disruption of intercellular junctions, the formation of multiple surface protrusions, and a rounded elevation of the normally flat cell body from the surface. 40 min of treatment with TPA, which was also associated with internalization of LAR and PTPσ on immunolabeling studies (Fig. 7 H), left the junctional morphology completely intact (Fig. 8 F). These observations indicated that only the EGTA- and ionophore-induced internalization was paralleled by a structural disruption of intercellular adhesions, whereas TPA-induced internalization occurred independently of this effect.

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