Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation, at any time, is the result of phosphatase and/or kinase activity. This balance is critical for control of growth and differentiation. The role of tyrosine phosphatases during nephrogenesis and in kidney disease requires delineation. Appropriate regulation of focal adhesion proteins such as focal adhesion kinase (FAK) and paxillin are important in cell adhesion, migration, and differentiation. We have previously shown that B cell lymphoma/leukemia-2 (bcl-2) −/− mice develop cystic kidneys and exhibit sustained phosphorylation of FAK and paxillin. We have examined the expression and activity of focal adhesion tyrosine phosphatases [Src homology-2 domain phosphatase (SHP-2), protein tyrosine phosphatase (PTP 1B), and PTP-proline, glutamate, serine, and threonine sequences (PEST)] during normal nephrogenesis and in cystic kidneys from bcl-2 −/− mice. Cystic kidneys from postnatal day 20 bcl-2 −/− mice demonstrate a reduced expression, sixfold decrease in activity, and altered distribution of SHP-2 and PTP 1B. PTP-PEST expression and distribution were similar in both bcl-2 +/+ and bcl-2 −/− mice. The altered regulation of PTP 1B and SHP-2 in kidneys from bcl-2 −/− mice correlates with sustained phosphorylation of FAK and paxillin. Thus renal cyst formation in the bcl-2 −/− mice may be the result of an inability of complete differentiation due to continued activation of growth processes, including activation of FAK and paxillin.
- focal adhesion kinase
- renal cysts
- focal adhesion tyrosine phosphatases
- protein tyrosine phosphatase-proline, glutamate, serine, and threonine sequences
- Src homology-2 domain phosphatase
- B-cell lymphoma/leukemia-2
renal hypoplasia/dysplasia/aplasiaare the second leading causes for renal transplantation in children. These renal diseases are the result of abnormal development caused by altered growth, differentiation, and/or organogenesis (29). The death repressor B cell lymphoma/leukemia-2 (bcl-2) plays an important role during nephrogenesis. Mice deficient in bcl-2 (bcl-2 −/−) develop renal hypoplasia/cystic dysplasia. Renal cyst formation in bcl-2 −/− mice, as well as other renal cystic diseases, is characterized by increases in apoptosis and proliferation, altered tubular morphology, and epithelial cells that do not fully differentiate (3, 4, 13, 23, 30, 31). Gross cyst formation in bcl-2 −/− mice occurs at multiple sites along the nephron at postnatal day 20 (P20) (25). We have observed nuclear localization of β-catenin, loss of apical brush-border actin staining, and sustained phosphorylation of focal adhesion kinase (FAK) and paxillin in cystic kidneys from bcl-2 −/− mice, suggesting that altered cell adhesive mechanisms occur (24, 26). Interestingly, renal cyst formation in these mice occurs at a time when bcl-2 expression normally has declined significantly. This suggests that early loss of bcl-2 during nephrogenesis may affect the ability of these kidneys to differentiate/mature at later times.
Tyrosine phosphatases play an important role during growth and differentiation. Altered regulation of tyrosine phosphatases may contribute to aberrant protein phosphorylation that leads to disease. The role these phosphatases play during nephrogenesis and renal cyst formation is unknown. We have recently demonstrated that FAK and paxillin are highly expressed and phosphorylated during normal nephrogenesis and decline to very low levels after renal maturation (P20) (26). In contrast, sustained phosphorylation of FAK and paxillin was observed in cystic kidneys fromP20 bcl-2 −/− mice. The overexpression of phosphorylated FAK or paxillin, in myoblasts, causes these cells to remain proliferative and inhibits their ability to initiate terminal differentiation (20); thus it is tempting to speculate that this occurs in cystic kidneys from bcl-2 −/− mice. Therefore, dephosphorylation of FAK and paxillin may play an important role during differentiation and renal maturation.
Src homology-2 domain phosphatase (SHP-2), protein tyrosine phosphatase (PTP 1B), and/or PTP-proline, glutamate, serine, and threonine sequences (PEST) can dephosphorylate focal adhesion proteins, such as FAK and paxillin, affecting cell growth and differentiation. Cells that lack expression of these phosphatases have hyperphosphorylation of FAK and paxillin as well as altered adhesion and migratory properties (2, 34). Cultured fibroblasts lacking functional SHP-2 have a reduced ability to spread and migrate on fibronectin, an increased number of focal adhesions, condensed F-actin aggregation at the cell periphery, and a significant reduction in FAK dephosphorylation (34). Cytoskeletal organization is altered similarly to that of FAK-deficient cells in these SHP-2 mutant cells. Cells deficient in PTP-PEST also have a phenotype similar to that of FAK-deficient cells, with altered cell migration and decreased turnover of focal adhesions. PTP-PEST acts to potentiate the action of FAK rather than antagonize it (2). These data suggest tyrosine phosphatases such as SHP-2 and PTP-PEST may work together with FAK to control the dynamics of focal adhesions (2, 34). Overexpression of PTP 1B in fibroblasts decreases cell migration, increases the time for cell spreading, and leads to formation of large focal complexes scattered over the ventral surface, which is similar to the reaction observed in PTP-PEST-deficient cells. Therefore, it is thought that an intermediate level of tyrosine phosphatase activity is necessary for formation of normal focal adhesions and cell migration (2).
To gain insight into the role SHP-2, PTP 1B, and PTP-PEST play during nephrogenesis and renal cyst formation, we have examined their expression, distribution, and activity in normal and cystic kidneys. Cystic kidneys from bcl-2 −/− mice displayed reduced expression and activity as well as an altered distribution of SHP-2 and PTP 1B. SHP-1 and PTP-PEST expression were not affected in these kidneys. Thus inappropriate expression and activation of tyrosine phosphatases SHP-2 and PTP 1B may impede terminal differentiation of renal epithelial cells, contributing to renal cyst formation.
MATERIALS AND METHODS
Bcl-2 heterozygote animals were interbred. The genotypes of the offspring were determined by PCR analysis, as previously described (26). To examine expression of SHP-2, SHP-1, PTP 1B, and PTP-PEST, normal mice were interbred and the kidneys were removed at the times noted. Embryos were removed from anesthetized pregnant female mice on day 15 of pregnancy. Kidneys were surgically dissected from embryos and postnatal mice. Samples from affected congenital polycystic kidney (cpk; cpk/cpk) mice and their normal littermates were generously supplied by Dr. James Calvet.
Protein lysate preparation and Western blot analysis.
Kidneys or metanephroi were homogenized and sonicated in a modified radioimmunoprecipitation assay buffer containing (in mM) 142.5 KCl, 5 MgCl2, 10 HEPES, pH 7.4, orthovanadate, and 2 sodium fluoride, as well as 1% Nonidet P-40 (NP-40) and a complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). The protein concentration was determined utilizing a Bio-Rad DC protein assay. Total protein lysate (20 μg) was electrophoresed on a 4–20% polyacrylamide gel and transferred to a Hybond enchanced chemiluminescence nitrocellulose membrane (Amersham, Arlington Heights, IL). The membranes were blocked for 1 h at room temperature in Tris-based saline buffer containing 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.05% Tween (TBST), 3% bovine serum albumin, and 3% nondairy creamer. The membranes were then incubated overnight at 4°C with rabbit polyclonal antibodies to PTP 1B, which recognize both p50 and p42 PTP 1B (1:1,000; Dr. Terry Woodford-Thomas, Washington University, St. Louis, MO) (30), rabbit polyclonal antibodies to PTP-PEST (amino acids 470–775: noncatalytic portion; 1:2,500; Dr. Michael Schaller, University of North Carolina, Chapel Hill, NC), or rabbit polyclonal antibodies to SHP-2 (syp; 1:1,500; Upstate Biotechnology, Lake Placid, NY). The membranes were then washed with TBST, incubated with the appropriate secondary antibody (Pierce, Rockford, IL), washed with TBST, and developed with enhanced chemiluminescence (Amersham).
Immunoprecipitation and phosphatase assays.
Kidneys were homogenized and sonicated in 150 mM NaCl, 50 mM Tris (pH 7.4), 1% NP-40, and protease inhibitors, but no phosphatase inhibitors. The protein concentration was determined, and 600 μg total protein were incubated with 3 μg anti-SHP-2, anti-PTP 1B, or IgG (as a negative control), followed by incubation with GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ). The immunoprecipitates were then washed three times in buffer containing 0.2% NP-40. The immunoprecipitates were incubated with 0.2 mM phosphopeptide RRLIEDAEpYAARG (Upstate Biotechnology) for 15 min. The phosphate released was measured by using malachite green (Upstate Biotechnology) according to the manufacturer's instructions. The reactions were linear over the time assayed.
Processing of kidneys for histological studies and immunohistochemistry.
Kidneys were surgically removed from mice, placed in an optimum cutting temperature compound (VWR Scientific, St. Louis, MO), and rapidly frozen. Sections of 7 μm each were placed on polylysine-coated slides (Sigma, St. Louis, MO). The sections were fixed in cold acetone (PTP 1B) or 3% paraformaldehyde (SHP-2 and PTP-PEST), washed in PBS, and incubated for 15 min in PBS blocking buffer (PBS containing 1% bovine serum albumin, 0.3% Triton X-100, and 0.2% skim milk powder). The sections were then incubated overnight at 4°C with rabbit polyclonal antibodies to PTP 1B (1:100; Upstate Biotechnology), SHP-2 (1:200; Upstate Biotechnology), or PTP-PEST (1:3,000; Dr. M. Schaller). The sections were incubated with indocarbocyanine (CY3)-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA). Nephron segments were identified by lectin staining and morphology. The sections were double stained with Lotus tetragonobolus agglutinin (Vector Laboratories, Burlingame, CA) to identify proximal tubules orDolichos biflorus agglutinin (Vector Laboratories) to identify collecting duct. For these experiments, fluorescein-labeled lectins (1:40) were incubated overnight with the primary antibody and processed as previously described (25). The slides were then photographed.
SHP-2 expression and activity are altered in cystic kidneys from bcl-2 −/− mice.
To understand the role tyrosine phosphatases have during differentiation and renal maturation, we have examined the expression, activity, and distribution of SHP-2. SHP-2 is a cytosolic tyrosine phosphatase that positively regulates cell differentiation and plays a role in growth factor- and integrin-signaling pathways. Phosphorylated SHP-2 is a 70-kDa protein (p70), and the dephosphorylated form is a 64-kDa protein (p64). SHP-2 can dephosphorylate FAK and other focal adhesion proteins, such as paxillin, thus modulating cell adhesion and migration (18). We examined SHP-2 expression in kidneys from embryonic day 15 (E15),P0, P10, and P20 mice to delineate expression during normal nephrogenesis and after renal maturation (atP20). In addition, we examined SHP-2 expression in cystic kidneys from P20 in bcl-2 −/− mice. At P20, gross cyst formation occurs at multiple sites along the nephron (25).
Phosphorylated p70 SHP-2 was highly expressed during normal nephrogenesis. In the normal kidney, dephosphorylated (p64) SHP-2 was virtually undetectable at E15 and P0. We first observed expression of p64 SHP-2 at P10, when renal maturation nears completion (Fig. 1). After renal maturation (P20) in kidneys from bcl-2 +/+ mice, there were significant amounts of both phosphorylated (p70) and unphosphorylated (p64) SHP-2 (Fig. 1). In contrast, p64 SHP-2 expression in kidney lysates from P20 bcl-2 −/− mice was low, which was similar to expression in normal P10 kidney lysates.
To determine the catalytic activity of SHP-2, kidney lysates fromP20 bcl-2 +/+, P20 bcl-2 −/−, andP10 bcl-2 +/+ mice were immunoprecipitated with anti-SHP-2 and the catalytic activity was determined, as described inmaterials and methods. The reactions were linear over the time assayed. SHP-2 released 1,408 ± 9 pmol phosphate/mg total protein from kidney lysates from P20 bcl-2 +/+ mice, 225 ± 3 pmol phosphate/mg total protein from P20 bcl-2 −/− mice, 165 ± 4 pmol phosphate/mg total protein fromP10 bcl-2 +/+ mice, and 100 ± 3 pmol phosphate/mg protein from lysates immunoprecipitated with IgG (negative control). Figure 2 is a graphic representation of the data, demonstrating a 6.3-fold decrease in SHP-2 activity in kidney lysates from P20 bcl-2 −/− mice compared with P20bcl-2 +/+ mice.
We have examined the distribution of SHP-2 in kidneys fromP20 bcl-2 +/+ (after renal maturation) and P20bcl-2 −/− mice (when significant cyst formation is observed). Nephron segments were identified by lectin staining and morphology. Kidneys from bcl-2 +/+ mice display intense SHP-2 staining on the cell periphery in inner medullary collecting ducts (Fig.3 G). No staining was observed in cortical or outer medullary collecting ducts (data not shown). In the cortex of the normal kidney, staining was observed in glomeruli (arrows, Fig. 3 A), distal tubules, and proximal tubules (Fig. 3 A). In proximal tubules, intense staining was noted on the apical membrane (arrowheads, Fig. 3 A). An altered distribution was observed in cystic kidneys from bcl-2 −/− mice. Kidneys from these mice displayed reduced staining in glomeruli (arrows, Fig. 3 B), loss of staining in cystic proximal tubules (arrows, Fig. 3 C), and reduced staining of the apical membrane of noncystic proximal tubules (arrowheads, Fig.3 B). Kidneys from bcl-2 −/− mice display intense SHP-2 staining in inner medullary collecting ducts that is similar to staining observed in bcl-2 +/+ mice (Fig. 3 H). Together, these results demonstrate that SHP-2 activity, expression, and distribution are altered in cystic kidneys from bcl-2 −/− mice.
Expression of a closely related tyrosine phosphatase, SHP-1, is not affected.
SHP-1 is a nontransmembrane tyrosine phosphatase with significant sequence identity to SHP-2 (16, 33). However, the two phosphatases have distinct biological roles (27). We have examined the expression of SHP-1 during nephrogenesis and in cystic kidneys from P20 bcl-2 −/− mice. SHP-1 was expressed at similar levels during nephrogenesis and in normal and cystic kidneys (Fig. 4). Because SHP-1 is constitutively expressed during nephrogenesis and SHP-2 expression increases after renal maturation in the normal kidney, these tyrosine phosphatases may have different functions during nephrogenesis.
PTP 1B expression and activity are altered in cystic kidneys from bcl-2 −/− mice.
PTP 1B is a 50-kDa tyrosine phosphatase that can be cleaved by calpain to a more catalytically active 42-kDa form (10). Both 42- and 50-kDa forms of PTP 1B are expressed in the kidney (Fig.5). P50 PTP 1B was highly expressed in kidneys from E15, P0, andP10 normal mice. Only a low level of p42 PTP 1B expression was observed in these lysates (Fig. 5). In kidneys from bcl-2 +/+ mice, expression of 50-kDa PTP 1B declined at renal maturation (P20), with a concomitant increase in 42-kDa PTP 1B expression (Fig. 5). In contrast, in kidney lysates from P20bcl-2 −/− mice, expression of PTP 1B remained low, which is similar to that observed in kidneys from normal P10 mice (Fig. 5).
We next determined the activity of PTP 1B in kidney lysates fromP20 bcl-2 +/+, P20 bcl-2 −/−, andP10 bcl-2 +/+ mice. Lysates were immunoprecipitated with anti-PTP 1B, and the phosphatase activity of the immunoprecipitates was determined. PTP 1B released 1,242 ± 10 pmol phosphate/mg total protein in kidney lysates from P20 bcl-2 +/+ mice, 225 ± 4 pmol phosphate/mg total protein from P20 bcl-2 −/− mice, 830 ± 8 pmol phosphate/mg total protein from P10 bcl-2 +/+ mice, and 115 ± 3 pmol phosphate/mg total protein from lysates immunoprecipitated with IgG (negative control). A graphic representation of the data, shown in Fig.6, demonstrates a 5.5-fold decrease in PTP 1B activity in kidney lysates from P20 bcl-2 −/− mice compared with P20 bcl-2 +/+ mice. Therefore, kidneys fromP20 bcl-2 −/− mice displayed a decreased expression of 42-kDa PTP 1B and decreased PTP 1B activity compared with their normal littermates.
The distribution of PTP 1B was examined in kidneys from P20bcl-2 +/+ and P20 bcl-2 −/− mice. In the normal adult kidney, PTP 1B was detected in distal tubules, collecting ducts, and glomeruli. Staining in distal tubules (arrow, Fig.7 A) and cortical (Fig.7 C) and medullary collecting ducts was observed on the cell periphery. Faint staining was also observed in proximal tubules (Fig.7 A). In contrast, cystic tubules in kidneys from bcl-2 −/− mice displayed altered localization and/or loss of PTP 1B expression. Figure 7 demonstrates that cystic medullary collecting ducts (arrowheads, Fig. 7 F) lose PTP 1B expression compared with noncystic collecting ducts (arrow, Fig. 7 F) in kidneys from bcl-2 −/− mice. In addition, in these kidneys, cystic distal tubules lose PTP 1B expression on the cell periphery and instead display apical brush border PTP 1B staining (arrowheads point to the apical membrane, Fig. 7 E). Noncystic distal tubules in these kidneys have a distribution of PTP 1B similar to that observed in bcl-2 +/+ mice (arrow, Fig. 7 E).
PTP-PEST expression is not affected in kidneys from bcl-2 −/− mice.
PTP-PEST is a tyrosine phosphatase also involved in regulating tyrosine phosphorylation of focal adhesion proteins. PTP-PEST binds signaling molecules, including Csk (8), paxillin (22), Shc (5, 14), and Grb2 (6), in its noncatalytic region. PTP-PEST is a 120-kDa protein that was highly expressed in kidney lysates from E15 normal mice (Fig.8). Expression of p120 PTP-PEST was observed at P0 but declined to very low levels at later postnatal times in these mice. The decline of p120 PTP-PEST occurred together with an increase in a 73-kDa protein (p73 PTP-PEST) during postnatal renal maturation. Expression of p120 and p73 PTP-PEST was similar in kidneys from P20 bcl-2 +/+ and P20bcl-2 −/− mice (Fig. 8).
The distribution of PTP-PEST was examined in kidneys fromP20 bcl-2 +/+ and P20 bcl-2 −/− mice. We observed a similar distribution of PTP-PEST in kidneys from bcl-2 +/+ and bcl-2 −/− mice. PTP-PEST was expressed in glomeruli, distal tubules, collecting ducts, thick ascending limbs, and proximal tubules (Fig. 9, A and B). We observed intense glomerular staining and focused staining on the apical surface of distal tubules (arrow, Fig. 9 A). In addition, proximal tubules display PTP-PEST staining, with nuclear staining particularly observed in the S2 and S3 segments (arrowheads, Fig. 9, A and B).
SHP-2 and PTP 1B expression is altered in cystic kidneys from cpk mice.
Affected cpk/cpk mice have polycystic kidney disease that is similar to autosomal-recessive polycystic kidney disease in humans (11, 15). Proximal tubules begin to dilate in embryonic and newborn animals, with the kidneys of the affected animals becoming grossly cystic during the first 3 wk of life. To determine whether the decreased expression of SHP-2 and PTP 1B was unique to cystic kidneys from bcl-2 −/− mice, we examined their expression in cystic kidneys from the affected cpk/cpk mice and normal littermates. We observed decreased expression of both these phosphatases in cystic kidneys from P20 bcl-2 −/− mice (Figs. 1 and 5). Figure 10 demonstrates Western analysis of protein lysates prepared from affectedP20 cpk/cpk (CY) mice and P20 normal littermates (NL) blotted for SHP-2 (A) and PTP 1B (B). We observed decreased expression of both SHP-2 and 42-kDa PTP 1B in lysates from the affected cpk/cpk mice compared with the normal littermates. The blots were stripped and reblotted with β-catenin to control for loading. We had previously observed that the expression of β-catenin is similar throughout nephrogenesis and in cystic kidneys (26). Similar expression of β-catenin was observed in cpk/cpk mice and normal littermates (data not shown).
Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation, at any time, is the result of kinase and/or phosphatase activity. Altered regulation of phosphatases and/or kinases can lead to abnormal growth and differentiation. The role of tyrosine phosphatases during nephrogenesis requires further delineation. We have examined the expression and activity of focal adhesion tyrosine phosphatases during normal nephrogenesis and in cystic kidneys from bcl-2 −/− mice. Our major findings are1) cystic kidneys display decreased expression and activity of SHP-2 and PTP 1B; 2) either loss or altered localization of SHP-2 and PTP 1B was observed in cystic kidneys; and 3) SHP-2 and PTP 1B are expressed with a nephron segmental distribution similar to FAK and paxillin in normal mice (Figs. 1 and 5). Therefore, aberrant regulation of these phosphatases may impede terminal differentiation of renal epithelial cells during renal maturation and thus facilitate renal cyst formation.
Cystic kidneys have increased amounts of apoptosis and proliferation, which suggests that epithelial differentiation is not complete (3, 4, 9, 12, 25, 31). These changes occur in the bcl-2 −/− mouse at a time when bcl-2 expression is normally low, suggesting that its loss early in development may alter signaling pathways, which ultimately results in the inability of these epithelial cells to terminally differentiate. In P20 bcl-2 −/− mice, renal cystic disease correlated with sustained phosphorylation of p125 FAK and p68 paxillin, although no changes in the levels of these proteins were observed (26). Because the overexpression of phosphorylated FAK or paxillin in myoblasts causes these cells to remain proliferative and inhibits their ability to initiate terminal differentiation (20), it is tempting to speculate that this occurs in cystic kidneys from the bcl-2 −/− mice. Our previous studies, as well as studies from others, indicate that downregulation of FAK and paxillin phosphorylation is essential for stabilization of focal adhesion complexes and promotion of a nonproliferative differentiated phenotype (20, 26). This is perhaps mediated, at least in part, through local recruitment and activation of focal adhesion tyrosine phosphatases. Therefore, appropriate dephosphorylation of these proteins may be required during nephrogenesis.
SHP-2 is a widely expressed cytosolic tyrosine phosphatase that plays a role in growth factor and integrin-mediated signaling pathways. SHP-2 can interact with various signaling molecules through its SH2 domains. It is recruited to focal adhesions and can modulate the activity of FAK (18). The loss of functional SHP-2 is lethal midgestation, with a phenotype similar to FAK- and fibronectin-deficient embryos caused by important migratory defects (21). The SHP-2 mutation also suppresses erythroid and myeloid differentiation, suggesting an important role during hematopoietic development (19). In addition, loss of functional SHP-2 in embryonic stem cells inhibits epithelial and fibroblast differentiation in vitro (19). Therefore, SHP-2 may positively mediate cell differentiation. In the studies presented here, expression and activity of SHP-2 were reduced in kidneys from bcl-2 −/− mice compared with their wild-type littermates. SHP-2 expression was also reduced in kidneys from the cpk/cpk mice. This would be consistent with epithelial cells from kidneys of P20 bcl-2 −/− andcpk/cpk mice, which express substantially lower amounts of SHP-2, being unable to fully differentiate. This altered expression appears to be specific for SHP-2, because SHP-1, a nontransmembrane tyrosine phosphatase with significant sequence identity to SHP-2 (16, 33), is not affected. These two phosphatases have distinct biological roles (27), which are apparent in these studies. Thus appropriate expression and activation of SHP-2 may be essential during normal nephrogenesis.
PTP 1B is a 50-kDa protein that has an ubiquitous tissue distribution. PTP 1B is cleaved by calpain to a more catalytically active 42-kDa protein (10). Introduction of wild-type PTP 1B into 3Y1 fibroblasts reduces phosphorylation of FAK and the rate of cell spreading (17). In addition, expression of dominant-negative PTP 1B results in a reduction of adhesion and spreading on fibronectin (1). Esophageal cancers also demonstrate a significant reduction of PTP 1B expression compared with surrounding tissue (28). The data presented here demonstrate a decrease in p42 PTP 1B expression and PTP 1B activity, as well as an altered distribution in cystic kidneys from P20bcl-2 −/− mice compared with P20 bcl-2 +/+ mice, thus suggesting a role for PTP 1B during renal maturation. We also observed decreased expression of 42-kDa PTP 1B in cpk/cpk kidneys. Together, these data indicate that altered expression of SHP-2 and PTP 1B may contribute to an immature cystic phenotype.
Tyrosine phosphatases localized to focal adhesion complexes are important in modulation of FAK activity during cell adhesion and migration. We believe that during the final stages of renal maturation, when downregulation of FAK and paxillin should occur, SHP-2 and PTP 1B are not properly recruited to focal adhesion complexes and/or activated. This hypothesis is supported by our data demonstrating decreased expression and activity and altered localization of PTP 1B in cystic kidneys. For example, in the normal kidney, PTP 1B localizes to the basolateral membrane where FAK and paxillin are expressed (26). However, in cystic distal tubules, PTP 1B is misdirected to the apical membrane; thus PTP 1B may not have access to FAK and paxillin to dephosphorylate the cystic distal tubules.
PTP-PEST is a cytoplasmic tyrosine phosphatase that can modulate focal adhesion complexes. It has a NH2-terminal catalytic domain and a COOH-terminal domain containing several PEST (8,22). Fibroblasts lacking PTP-PEST have decreased migration, increased number of focal adhesions, and hyperphosphorylation of FAK and paxillin (7). We observed similar levels and distribution of PTP-PEST in kidneys from P20 bcl-2 −/− andP20 bcl-2 +/+ mice. At P20, p73 PTP-PEST is the most abundant form, and is recognized by an antibody against the noncatalytic domain. Thus PTP-PEST expression is similar in kidneys from bcl-2 +/+ and bcl-2 −/− mice.
In summary, the appropriate recruitment and activation of tyrosine phosphatases to focal adhesion complexes are essential for stabilization of cellular adhesive mechanisms and modulation of cell adhesion and migration. The decreased activity and expression, as well as an altered distribution of PTP 1B and SHP-2, are consistent with sustained phosphorylation of FAK and paxillin and the immature/undifferentiated cystic phenotype in bcl-2 −/− mice. Our data demonstrating that SHP-2 and PTP 1B expression is reduced in cystic kidneys from the cpk mice suggest that these changes are not unique to the bcl-2 −/− mouse. Thus proper regulation of tyrosine phosphatases, including SHP-2 and PTP 1B, may play an important role during renal maturation.
The authors thank Dr. Anna Huttenlocher for critically reviewing the manuscript and Drs. Terry Woodford-Thomas and Michael Schaller for generously supplying antibodies for PTP 1B and PTP-PEST, respectively. We greatly apppreciate the generosity of Dr. James Calvet for supplying us with samples from the affected cpk/cpk mice and their normal littermates.
This research (and C. M. Sorenson) was funded by a Scientist Development Grant from the American Heart Association and a Solomon Papper MD Young Investigator Grant from the National Kidney Foundation. N. Sheibani was funded by National Institutes of Health Grant AR-45599.
Address for reprint requests and other correspondence: C. M. Sorenson, Dept. of Pediatrics, Univ. of Wisconsin-Madison, H4/444 CSC, 600 Highland Ave., Madison, WI 53792–4108 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2002 the American Physiological Society