Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293  cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LARi, or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP  LARi-glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin (A) or plakoglobin (B). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141638&req=5

Figure 11: In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293 cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LARi, or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP LARi-glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin (A) or plakoglobin (B). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.

Mentions: To determine whether the PTPases not only localize to adherens junctions and desmosomes but can also associate with known proteins of the cell adhesion complexes at these sites, we performed in vitro association experiments. E-cadherin, α- and β-catenin, and plakoglobin were transiently expressed in 293 cells. Cells were incubated with or without the phosphatase inhibitor pervanadate before lysis to study the influence of tyrosine phosphorylation of these proteins on the association with LAR. Lysates were then incubated with glutathione-sepharose–bound GST-fusion protein, GST–hPTP-LARi, containing the entire intracellular domain of LAR, from amino acids 1,259 to 1,881. Bound proteins were analyzed by immunoblotting with antibodies directed against β-catenin (Fig. 11 A), plakoglobin (Fig. 11 B), α-catenin (data not shown), and E-cadherin (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)

In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293  cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LARi, or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP  LARi-glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin (A) or plakoglobin (B). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141638&req=5

Figure 11: In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293 cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LARi, or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP LARi-glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin (A) or plakoglobin (B). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.
Mentions: To determine whether the PTPases not only localize to adherens junctions and desmosomes but can also associate with known proteins of the cell adhesion complexes at these sites, we performed in vitro association experiments. E-cadherin, α- and β-catenin, and plakoglobin were transiently expressed in 293 cells. Cells were incubated with or without the phosphatase inhibitor pervanadate before lysis to study the influence of tyrosine phosphorylation of these proteins on the association with LAR. Lysates were then incubated with glutathione-sepharose–bound GST-fusion protein, GST–hPTP-LARi, containing the entire intracellular domain of LAR, from amino acids 1,259 to 1,881. Bound proteins were analyzed by immunoblotting with antibodies directed against β-catenin (Fig. 11 A), plakoglobin (Fig. 11 B), α-catenin (data not shown), and E-cadherin (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