Interaction of bcl-2 with Paxillin through Its BH4 Domain Is Important during Ureteric Bud Branching

bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Mice deficient in bcl-2 (bcl-2-/-) develop renal hypoplastic/cystic dysplasia, a condition that leads to significant morbidity and mortality in children. The precise mechanism of action of bcl-2 has not been elucidated. bcl-2 may merely facilitate survival of precursor cells and/or may play a more "active" role during morphogenesis by interacting with other proteins such as paxillin. Recent work in this laboratory demonstrated that bcl-2 directly associates with paxillin. The data presented here demonstrate that the bcl-2 homology 4 (BH4) domain, specifically amino acids 17-31, is necessary for the bcl-2 interaction with paxillin. Paxillin also associated with the BH4 domains of more closely related bcl-2 family members, bcl-xL and bcl-w, compared with that from the non-mammalian homologue ced9. Tyrosines 21 and 28 in the bcl-2 BH4 domain were essential for interaction with paxillin. In embryonic kidney organ culture, incubation with the bcl-2 BH4 domain resulted in inhibition of ureteric bud branching. Therefore, these data suggest that the interaction of bcl-2 with paxillin plays an important role during nephrogenesis.

bcl-2 is a novel proto-oncogene that inhibits apoptosis rather than promoting proliferation. Overexpression of bcl-2 increases the viability of cells under various adverse circumstances including cytokine withdrawal, loss of cell adhesion, and ␥-irradiation. bcl-2 is inserted into the outer mitochondrial, endoplasmic reticulum, and nuclear membranes in an N cyto -C in orientation with the bulk of the protein facing the cytosol (1). bcl-2 contains four conserved domains denoted bcl-2 homology (BH) 1 1 (residues 136 -155), BH2 (residues 187-202), BH3 (residues 93-107), and BH4 (residues 10 -30) as well as a transmembrane-spanning region at the carboxyl end of the protein (2). Through the BH domains bcl-2 forms homo-and heterodimers with various bcl-2 family members impacting the regulation of apoptosis.
The BH4 domain is conserved in bcl-2 family members with death-repressing activity. bcl-2 with the BH4 domain deleted (amino acids 10 -30) lacks the survival activity (3). Removal of the amino-terminal portion of bcl-2 by cleavage by caspase 3 or trypsin-like protease results in a carboxyl fragment that can accelerate cell death (4,5). The BH4 domain interacts with non-family members including Raf-1 kinase, calcineurin, and ced4 (3,6,7). Unfortunately, the mechanism by which bcl-2 imparts its death-inhibitory activity and whether bcl-2 functions in other capacities require further investigation.
bcl-2 expression can be regulated by interaction of integrins with components of the basement membrane. For example, overexpression of bcl-2 in epithelial cells promotes dedifferentiation, a loss of expression of E-cadherin and ␣2␤1 integrin, and multilayer formation at high density (8). bcl-2 transcription is elevated in cells attached to fibronectin through ␣5␤1 or vitronectin through ␣v␤3 (9). Regulation of cell survival function by bcl-2 requires FAK, Shc, and activated Ras (through phosphatidylinositol 3-kinase/Akt pathway) (9).
Recent work in this laboratory demonstrated that bcl-2 coimmunoprecipitated with FAK and paxillin in embryonic kidney lysates (10). Utilizing Far Western analysis, the interaction between bcl-2 and paxillin was shown to be direct, whereas its interaction with FAK was indirect. Gaining a better understanding of the molecular and cellular pathways by which bcl-2 mediates cell survival is essential for determining its role during development. Here the association between bcl-2 and paxillin was further examined, demonstrating that the BH4 domain, specifically amino acids 17-31, is necessary for the bcl-2 interaction with paxillin. Paxillin also associated with the BH4 domains of more closely related bcl-2 family members, bcl-xL and bcl-w, compared with that from the non-mammalian homologue ced9. Tyrosines 21 and 28 in murine bcl-2 were essential for interaction with paxillin. In embryonic kidney organ culture, incubation with the bcl-2 BH4 domain resulted in inhibition of ureteric bud branching and morphogenesis. In contrast, incubation of embryonic kidneys with a mutant bcl-2 BH4 Y21D/Y28D peptide did not affect kidney morphogenesis. Therefore, the association of paxillin with bcl-2 BH4 domain may play a role during the early stages of nephrogenesis impacting ureteric bud branching.

MATERIALS AND METHODS
Transfection with Deletion Constructs-293 cells (human embryonic kidney cells) were transfected, using Lipofectin (Invitrogen), with murine bcl-2 deletion constructs in pcDNA3.1(Ϫ)/MycHisA (tag in-frame with stop codon) (Invitrogen) containing 1-90 amino acids (aa), 1-140 aa, 1-190 aa, or vector (as a control). The inserts for the bcl-2 deletion constructs were prepared by PCR using the following primers: bcl-2 purified using a Spin Q column (Qiagen, Vaencia, CA), digested with NheI and EcoRV, ligated into the pcDNA3.1(Ϫ)/MycHisA vector cut with the same enzymes, and transformed into Escherichia coli DH5␣. Bacterial colonies were screened by NheI and EcoRV digestion of minipreps, and those with inserts of the appropriate size were sequenced using the Big Dye reagent (PerkinElmer Life Sciences) as described by the manufacturer. The samples were analyzed by the DNA sequencing facility at the University of Wisconsin Biotechnology Center (Madison, WI). The transfectants were grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum in the presence of 500 g/ml G418. A population of transfectants was immunoprecipitated with anti-myc (9E10; Sigma) and Western blotted for paxillin (BD Biosciences) essentially as described previously (10).
Peptide Binding Assay-The nickel-nitrilotriacetic acid-agarose (200 l; Qiagen) was washed with Hepes-buffered salt solution (20 mM Hepes, 150 mM NaCl, pH 7.4), and 1-2 mg of the specific His 6 -tagged peptide was bound to the agarose. The peptides described in Table I were synthesized at the University of Wisconsin Biotechnology Center with or without His 6 on the amino terminus. These peptides were used in the experiments described under "Results." As a source of protein NIH3T3 cells were chosen because they are mouse cells that express a significant amount of paxillin. NIH3T3 cell lysates were prepared in a modified radioimmune precipitation assay buffer containing 142.5 mM KCl, 5 mM MgCl 2 , 10 mM Hepes, pH 7.4, 1% Nonidet P-40, and Complete protease inhibitor mixture (Roche Applied Science). The protein concentration was determined using a Bio-Rad DC protein assay. 500 g of NIH3T3 lysate was incubated for 2 h with the peptide of interest bound to Ni-agarose (Qiagen) at 4°C and then washed three times with radioimmune precipitation assay buffer containing 10 mM imidazole. A portion of the sample was Western blotted for paxillin. To analyze for paxillin-associated proteins, a portion of the sample was Western blotted using the following antibodies: anti-ILK (Upstate Biotechnology, Lake Placid, NY), antivinculin (Sigma), anti-PTP-PEST (provided by Dr. M. Schaller), anti-Csk, anti-Crk, and anti-FAK (BD Biosciences).
Embryonic Kidney Organ Culture-Embryos were removed from timed pregnant FVBN mice at embryonic day 11.5-12, when the ureteric bud was in a T-shape. Embryonic kidneys were surgically dissected from the embryos. To minimize the variability between kidneys originating from different embryos, comparisons were made between kidneys from the same embryo (right and left kidneys) (11,12). Cultures of embryonic kidneys were carried out on a Transwell clear polyester membrane (0.4 m; Costar) using Dulbecco's modified Eagle's medium:F12 medium (Invitrogen) supplemented with 5ϫ MITO (BD Biosciences), 50 units/ml penicillin, 50 g/ml streptomycin (Sigma), 50 g/ml gentamicin (Invitrogen), and 50 units/ml nystatin (Sigma). For some experiments embryonic kidneys were incubated with vehicle or the following TAT-labeled peptides (20 M) for 4 days at 37°C: bcl-2 BH4 7-31 -TAT peptide (DNREIVMKYIHYKLSQRGYEWDYGRKKR-RQRRRG), bcl-2 BH4 17-31 -TAT (KYIHYKLSQRGYEWDYGRKKRR-QQRRRG), or PECAM-1 exon 14-TAT (LGTRATETVYSEIRKVD-PYGRKKRRQRRRG). For other experiments, 20 M untagged or 10 M His 6 -tagged peptides were incubated with the Chariot reagent (Active Motif, Carlsbad, CA), and medium was added. All peptides were puri-fied and dissolved in water prior to use. The photomicrographs were taken, and medium (with or without peptide) was changed daily.
In some cases the embryonic kidneys were processed for histological studies. Embryonic kidneys were placed in OCT (VWR Scientific, St. Louis, MO) and rapidly frozen. Sections of 7 m each were placed on polylysine-coated slides and stained with hematoxylin-eosin (H&E). The percentage of pyknotic nuclei were also scored by counting 200 cells from representative areas in kidney sections.
To localize the ureteric bud, whole mount staining of embryonic kidneys with fluorescein isothiocyanate-labeled Dolichos biflorus agglutinin (Vector, Burlingame, CA) was performed. The embryonic kidneys were fixed overnight in 2% paraformaldehyde and blocked in 50 mM NH 4 Cl. The embryonic kidneys were incubated for 30 min with 0.075% saponin and then 0.075% saponin with 0.2% gelatin. The embryonic kidneys were next incubated with the saponin-gelatin solution containing D. biflorus agglutinin (1:80) for 1 h at room temperature, washed in phosphate-buffered saline, and placed on coverslips. Images were obtained using a Zeiss microscope (AxioPhot, Carl Zeiss, Chester, VA) equipped with a digital camera (Axiovision, Carl Zeiss).

Paxillin Interacts with an Amino-terminal Fragment of bcl-2-
The molecular and cellular pathways by which bcl-2 mediates cell survival are not clearly understood. Apoptosis controls inappropriate cell positioning by requiring that differentiated epithelial cells remain in contact with their matrix for survival. Overexpression of bcl-2 protects cells from apoptosis caused by loss of adhesion. However, the mechanism by which bcl-2 protects cells as a result of loss of adhesion remains to be determined. I hypothesized that bcl-2 functions in part by interacting with paxillin. Support for the idea comes from my observation that bcl-2 and paxillin co-immunoprecipitate in embryonic kidney protein lysates. This interaction was shown to be direct by Far Western analysis (10).
To identify the domain in bcl-2 that interacts with paxillin, 293 cells were transfected with bcl-2 deletion constructs in pcDNA3.1(Ϫ)/MycHisA (tag in-frame with stop codon). These constructs contained 1-90, 1-140, or 1-190 aa of bcl-2 (description of constructs is shown in Fig. 1A). Stable transfectants were obtained. The vector without an insert served as a control. The resulting cell populations were immunoprecipitated with anti-myc and Western blotted for paxillin. Fig. 1B demonstrates that paxillin co-immunoprecipitated with the first 90 aa of bcl-2 but not with the vector alone. I also observed association between paxillin and the other bcl-2 deletion constructs described in Fig. 1A (data not shown). Thus, expression of the first 90 amino acids of bcl-2 was sufficient for association with paxillin.
Paxillin Interacts with the bcl-2 BH4 Domain-Located Peptides with or without a His 6 on the amino terminus bcl-2 BH4-(7-31) LGTRATETVYSEIRKVDPYGRKKRRQRRRG bcl-2, Paxillin, and Ureteric Bud Branching within the first 90 amino acids of bcl-2 is the BH4 domain (amino acids 10 -30). To determine whether the BH4 domain was sufficient for association with paxillin, a His 6 -tagged bcl-2 BH4 domain peptide (amino acids 7-31) was synthesized. The His 6 -tagged bcl-2 BH4 peptide bound to nickel-agarose was incubated with NIH3T3 cell lysates, and the eluted protein(s) were Western blotted for paxillin. Fig. 2A demonstrates that the bcl-2 BH4 domain binds paxillin. 30 g of NIH3T3 protein lysate and a comparable amount from the original starting material bound to BH4-or BH2-agarose was Western blotted for paxillin. Approximately 75% of paxillin in NIH3T3 cell lysates binds to the BH4 domain peptide (compare 3T3 lysate to BH4 lanes) under these experimental conditions ( Fig. 2A). Furthermore, paxillin did not associate with a His 6 -tagged bcl-2 BH2 domain peptide ( Fig. 2A), demonstrating that association between paxillin and bcl-2 was specific for the BH4 domain. Addition of excess untagged BH4 peptide decreased the amount of paxillin associating with the His 6 -tagged bcl-2 BH4 peptide (Fig. 2B).
Paxillin Interacts with the BH4 Domain from Other bcl-2 Family Members-To narrow the portion of the bcl-2 BH4 domain that was essential for paxillin binding, I next synthesized His 6 -tagged peptides to bcl-2 amino acids 7-17 and 17-31. Fig. 3 demonstrates that paxillin associated with amino acids 17-31 but not amino acids 7-17 from the bcl-2 BH4 domain (Fig. 3, A and B). The next question asked was what amino acids were critical for binding. Instead of mutating each amino acid individually to determine which one(s) were important, the diversity of the BH4 domains within the bcl-2 family members was exploited. Mammalian as well as non-mammalian (ced9 and balf1) bcl-2 family members contain BH4 domains. Although some similarity exists between these do-mains, they are not identical (Fig. 3C). His 6 -tagged peptides to the matching amino acids 17-31 in the bcl-2 BH4 domain from bcl-xL, bcl-w, balf1, and ced9 were synthesized. The sequences for the domain with the actual peptides synthesized and association with paxillin are described in Fig. 3C. Paxillin associated with the BH4 domains of more closely related bcl-2 family members, bcl-xL and bcl-w. Some binding of paxillin to the balf1 BH4 domain was observed, but little if any association was observed with another non-mammalian homologue, ced9 (Fig. 3).
Substitutions in the bcl-2 BH4 Domain Affect Association with Paxillin-Paxillin associated with closely related mammalian bcl-2 family members and to a lesser degree with nonmammalian homologues (Fig. 3). The amino acid sequence differences between the BH4 domains of the bcl-2 family members were examined and compared with their ability to associate with paxillin. Next, peptides were synthesized with substitutions that could potentially affect binding to paxillin. The initial substitutions made were S24D, K22A/R26A, and Y21D/ Y28D. The binding assays were performed as described under "Materials and Methods." Substituting S24D and K22A, R26A did not affect the association of paxillin with the bcl-2 BH4 domain (Fig. 4A). In contrast, a phosphorylation mimenic substitution (Asp) for Tyr-21 and Tyr-28 (7 amino acids apart) blocked paxillin binding. Even when a more conservative substitution for the tyrosines was made (Tyr 3 Phe) association with paxillin did not occur (Fig. 4B). The inability of paxillin to bind to the BH4 domain when tyrosines 21 and 28 are substituted with phenylalanine or aspartic acid could explain why no significant association between paxillin and the ced9 BH4 domain, which lacks these tyrosines, was observed. To address this possibility the ced9 BH4 domain was synthesized with histidine 90 and methionine 97 replaced with tyrosines. As shown in Fig. 4B, changing amino acids 90 and 97 to tyrosines was sufficient to facilitate paxillin association with the ced9 BH4 domain. Thus, these data indicate that the unphosphorylated tyrosines spaced 7 amino acids apart in the bcl-2 BH4 domain may play a role in the association with paxillin. These data are summarized in Fig. 4C. Even the seemingly minor substitution Y21F/Y28F may cause significant topographical changes in the BH4 structure such that association with paxillin is inhibited.

bcl-2, Paxillin, and Ureteric Bud Branching
Paxillin Interacting with bcl-2 Does Not Affect Its Association with Other Proteins-To examine whether other proteins that normally bind paxillin can associate when paxillin binds the bcl-2 BH4 domain, the His 6 -tagged BH4 peptide bound to nickel-agarose was incubated with NIH3T3 cell lysates. Western blotting for several paxillin-associated proteins was performed. ILK, PTP-PEST, Crk, Csk, FAK, and vinculin associated with bcl-2 BH4-bound paxillin (Fig. 5).
Incubation with the BH4 Domain Peptide Inhibits Ureteric Bud Branching-To begin to examine the role the bcl-2 BH4 domain plays in vivo, embryonic kidney (metanephric) organ culture was utilized. Embryonic kidneys grown in organ culture undergo the complex early events in renal differentiation in a manner similar to that observed in the embryo. My laboratory previously used this model system to examine abnormal kidney development in bcl-2Ϫ/Ϫ and Os/ϩ mice (11,12). To minimize the variability between kidneys originating from different embryos, comparisons were made between kidneys from the same embryo (right and left kidneys).
Embryonic kidneys from embryonic day 11.5 (E11.5) pregnant normal mice were grown in the presence of vehicle or 20 M BH4 bcl-2-TAT peptide (DNREIVMKYIHYKLSQR-GYEWDYGRKKRRQRRRG) for 4 days at 37°C. To ensure penetration of the peptide I linked a highly basic peptide derived from the human immunodeficiency virus TAT protein (YGRKKRRQRRRG) to the carboxyl terminus. This sequence is sufficient to enable translocation of peptides across cell membranes (13,14). Fig. 6A demonstrates a photomicrograph and corresponding H&E sections of embryonic kidneys incubated in the presence or absence of 20 M BH4 bcl-2-TAT peptide. After 4 days in organ culture, growth was noticeably impaired in embryonic kidneys incubated with the BH4 bcl-2-TAT peptide. Next, H&E sections from these embryonic kidneys were examined. Components of the ureteric bud and surrounding metanephric blas-

FIG. 3. Paxillin associates with the BH4 domains from bcl-2 family members bcl-w and bcl-xL.
In A, His 6 -tagged bcl-2 amino acids 7-17 or amino acids 7-31 (BH4) or BH2 domain peptide bound to nickel-agarose was incubated with NIH3T3 cell lysate. The eluted protein(s) were Western blotted for paxillin. B demonstrates similar experiments utilizing the BH4 domain from bcl-2 family members. C indicates the peptides used and summarizes their general ability to associate with paxillin. These experiments were repeated three times with similar results.

FIG. 4. Amino acid substitutions in the BH4 domain.
Peptides were synthesized that contained substitution(s) that would potentially affect association with paxillin. In A, S24D, K22A/R26A, and Y21D/ Y28D substitutions were made in the bcl-2 BH4 domain. Binding assays were performed. Note that a phosphorylation mimenic substitution (Asp) for Tyr-21 and Tyr-28 (7 amino acids apart) blocked paxillin binding. In B, the role of the tyrosines spaced 7 amino acids apart was further investigated. bcl-2 BH4 peptides containing a Y21F/Y28F substitution and a replaced ced9 BH4 domain (histidine 90 and methionine 97 replaced with tyrosines) were used in binding assays. C indicates the peptides used and summarizes their general ability to associate with paxillin. These experiments were repeated twice with similar results. bcl-2, Paxillin, and Ureteric Bud Branching tema were present in both control and BH4 peptide-incubated embryonic kidneys. Although tubulogenesis occurred in the BH4 bcl-2-TAT peptide-incubated embryonic kidneys, it was markedly reduced. To determine whether cell viability declined in the presence of the BH4 peptide pyknotic nuclei were scored. A 3-fold increase in pyknotic cells (3% in controls versus 9% in BH4-incubated) was observed in embryonic kidneys incubated with the BH4 peptide.
To visualize the ureteric bud, embryonic kidneys were whole mount-stained with D. biflorus agglutinin (Fig. 6B). Embryonic kidneys incubated with the bcl-2 BH4 domain exhibited substantially less ureteric bud branching. The embryonic kidneys also appear slightly smaller than control kidneys. I repeated these experiments with bcl-2 BH4 17-31 -TAT peptide with similar results (data not shown). This was not merely due to the presence of the TAT peptide because incubation of embryonic kidneys with a PECAM-1 exon 14-TAT peptide did not affect ureteric bud branching or kidney morphogenesis. To further ensure that the observed effect was attributable to the bcl-2 BH4 domain, I repeated these experiments using the Chariot reagent to deliver untagged bcl-2 BH4 peptide to the embryonic kidneys. Similar results were observed as those shown with the BH4 TAT-labeled peptides (data not shown). As an additional control, the BH4 Y21D/Y28D-mutated peptide was used. The BH4 Y21D/Y28D peptide did not associate with paxillin in the binding experiments performed above (Fig. 4). Morphogenesis and ureteric bud branching were similar in control and BH4 Y21D/Y28D-mutated peptide-incubated embryonic kidneys. These data suggest that both binding of the bcl-2 BH4 domain to paxillin as well as the inhibitory effect of the BH4 peptide in embryonic kidney organ culture require tyrosines 21 and 28 in bcl-2 BH4 domain.
The next question asked was whether paxillin associated with the BH4 peptide in embryonic kidneys incubated in organ culture. The Chariot protein delivery agent was used to deliver the His 6 -tagged bcl-2 BH4 domain peptide. This lipid-based protein delivery system efficiently translocates macromolecules into cells. E11.5 embryonic kidneys were grown on filters for 20 h. The medium contained either Chariot alone (control) or Chariot with the 10 M His 6 -tagged bcl-2 BH4 peptide. Protein lysates prepared from the embryonic kidneys were incubated with nickel-agarose, and the bound proteins were analyzed by Western blot analysis. Fig. 7 shows that only lysates from His 6 -tagged bcl-2 BH4 peptide-incubated embryonic kidneys demonstrate association with paxillin. Thus, it appears that the peptide is taken up by the embryonic kidneys and associates with paxillin under these experimental conditions.

DISCUSSION
Apoptosis plays a critical role during development and in the maintenance of multicellular organisms. bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion (8,9). bcl-2 may facilitate survival of precursor cells or play a more active role during morphogenesis by interacting with other proteins. Gaining a better understanding of the molecular and cellular pathways by which bcl-2 mediates cell survival is essential for determining its role during devel-opment. bcl-2 co-immunoprecipitates with paxillin and FAK in lysates from embryonic kidneys (10). Utilizing Far Western analysis, the interaction between bcl-2 and paxillin was shown to be direct, whereas its interaction with FAK was indirect. Here, the association between bcl-2 and paxillin has been investigated further. The major findings are as follows. 1) The bcl-2 BH4 domain, specifically amino acids 17-31, interact with paxillin. 2) Mutating tyrosines 21 and 28 in the BH4 domain of bcl-2 abolishes binding to paxillin. Tyrosines at these locations are conserved in the more closely related bcl-2 family members bcl-w and bcl-x. 3) Replacing histidine 90 and methionine 97 in the ced9 BH4 domain with tyrosines facilitated association with paxillin. 4) Incubation of embryonic kidneys in organ culture with a BH4 domain peptide inhibits ureteric bud branching and morphogenesis. In contrast, incubation of embryonic kidneys with a BH4 Y21D/Y28D peptide does not significantly affect ureteric bud branching. Therefore, bcl-2 interaction with paxillin through its BH4 domain may be essential during nephrogenesis.
Paxillin is a 68-kDa focal adhesion protein that interacts with focal adhesion and signal transduction proteins. The paxillin amino terminus contains binding sites (leucine-rich motif; LD motifs) for FAK, Csk, Src, PYK2, and vinculin (15)(16)(17)(18)(19), while the carboxyl terminus has four LIM domains targeting it to focal adhesions. Paxillin is required during embryonic development and acts as a cytoplasmic effector for some fibronectin receptors (20). Moreover, fibronectin expression is important for cleft formation during branching morphogenesis in the salivary gland (21). Paxillin-deficient mice are abnormal by E8 (prior to kidney development) (20,22). Paxillin can function during the regulation of migration, cell spreading, and phosphorylation. This may be the direct or indirect result of interaction with proteins such as FAK, Crk, Cas, PTP-PEST, and Csk. ILK, PTP-PEST, Crk, Csk, FAK, and vinculin associated with bcl-2 BH4 peptide-bound paxillin (Fig. 5). Thus, targeting paxillin to specific regions of a particular organ during development could have profound implications as to the microcosm of signaling molecules available in that area.
bcl-2 is widely expressed during development and becomes restricted upon maturation in many tissues. The kidney demonstrates this quite well. During nephrogenesis apoptosis tends to be inversely correlated with bcl-2 expression (23). bcl-2 is highly expressed early in the embryonic kidney, and its level decreases significantly at later times such that its expression is normally low in the postnatal kidney (24). In the developing kidney bcl-2 is expressed in the ureteric bud (24) and in epithelial condensates of the metanephric blastema (23). However, bcl-2 is not expressed in uninduced mesenchyme (23). Loss of bcl-2 dramatically affects kidney development. Mice deficient in bcl-2 (bcl-2Ϫ/Ϫ) develop renal hypoplasia/cystic dysplasia. Kidneys in these mice undergo fulminant apoptosis of the metanephric blastema during early embryogenesis (E12), and the renal epithelial cells do not complete terminal differentiation in the postnatal kidney (10,12,(25)(26)(27)(28). Perhaps early embryonic expression of bcl-2 and its association with paxillin facilitate morphogenesis by supporting survival of precursor cells allowing them to be less adherent and more migra- bcl-2, Paxillin, and Ureteric Bud Branching tory without the threat of apoptosis, thus allowing them to differentiate appropriately.
Proper regulation of apoptosis is essential for nephrogenesis to proceed normally. Although bcl-2 is known to be highly expressed in the murine kidney following induction, its normal function is not completely understood. Loss of bcl-2 impairs ureteric bud branching and causes fulminant apoptosis of the metanephric blastema (12). 2 Whether bcl-2 merely acts as a survival factor or plays a more active role by associating with 2 C. M. Sorenson, unpublished data. paxillin as part of a differentiation cascade during nephrogenesis remains to be seen. Enhanced cell survival has two consequences. The obvious one is that it maintains the progenitor cell population. The less obvious one is that it allows progenitor cells to disengage from the substratum and to migrate, resulting in the formation of nephron segments. The notion that bcl-2 can influence cell migration is supported by the fact that bcl-2 expression is regulated by interaction of integrins with components of the basement membrane (9,29). Thus, the early embryonic expression of bcl-2 and its association with paxillin could facilitate morphogenesis by supporting survival of precursor cells allowing them to be less adherent and therefore more able to migrate without the threat of apoptosis. This is consistent with the fulminant apoptosis observed in embryonic kidney from bcl-2Ϫ/Ϫ mice (12).
The BH4 domain is found in most bcl-2 family members with death-repressing activity. It is most conserved between mammalian family members (bcl-2, bcl-x, and bcl-w) and less conserved between non-mammalian family members (ced9 and balf1). The bcl-2 BH4 domain is thought to be sufficient and necessary for the cell survival activity of bcl-2 (3). bcl-2 with the BH4 domain deleted has been reported to lack survival function but still avidly binds to bax, bik/nbk, bak, bad, bid, and bim with a similar affinity as wild-type bcl-2 (3). Caspase cleavage of bcl-2 yields a pro-apoptotic 23-kDa bcl-2 fragment that has lost its BH4 domain (4). Therefore, the BH4 domain appears to mediate the anti-apoptotic activity of bcl-2 independent of its interactions with other bcl-2 family members.
Paxillin associated with the bcl-2 BH4 domain and BH4 domains of closely related bcl-2 family members (bcl-w and bcl-xL). This association required tyrosines 21 and 28 in the bcl-2 BH4 domain (7 amino acids apart). Substituting these tyrosines to either phenylalanine or aspartic acid inhibited association with paxillin. The ced9 BH4 domain, which lacks these tyrosines, did not demonstrate significant association with bcl-2. Substituting other residues that would influence BH4 domain charge, such as S24D and K22A/R26A, did not affect the association with paxillin. Furthermore, association of paxillin with the ced9 BH4 domain was restored by changing amino acids 90 and 97 to tyrosines. Thus, even a seemingly minor Y21F/Y28F substitution may cause significant topographical changes in the BH4 structure such that association with paxillin is prohibited.
Exogenous addition of BH4 domain to embryonic kidneys acts in a dominant negative fashion, disrupting kidney development. Although components of the ureteric bud and surrounding metanephric blastema were present in both control and BH4 peptide-incubated embryonic kidneys, incubation with the bcl-2 BH4 domain noticeably impaired ureteric bud branching and tubulogenesis. Cell death is also increased in embryonic kidneys incubated with the BH4 peptide. The BH4 domain-incubated embryonic kidneys appear similar to embryonic kidneys from bcl-2Ϫ/Ϫ mice. The importance of the interaction of bcl-2 with paxillin is underscored by the inability of embryonic kidneys incubated with bcl-2 BH4 Y21D/Y28D peptide to affect ureteric bud branching. It is tempting to speculate that the association of bcl-2 with paxillin supports branching morphogenesis by circumventing the need for integrin signaling for survival. An alternative explanation is that the bcl-2 interaction with paxillin recruits other important players in integrin signal transduction that associate with paxillin, positively influencing differentiation. Therefore, disruption of ureteric bud branching may be most sensitive to the BH4 domain early when bcl-2 expression is high and before the signaling machinery orchestrating branching is firmly in place.
The role bcl-2 plays as a survival factor appears to be rather complex, perhaps utilizing different pathways for specific purposes. For example, the manner in which bcl-2 facilitates survival of mesenchyme and ureteric bud cells during nephrogenesis may not be identical. The mode of morphogenesis and the organ itself may predetermine the method by which bcl-2 or other family members act as survival factors. Therefore, gaining a better understanding of the interrelationship between cell survival and adhesive processes will further our insight into nephrogenesis. FIG. 7. Association of paxillin with the BH4 peptide in embryonic kidneys in organ culture. The Chariot protein delivery agent was used to deliver the His 6 -tagged bcl-2 BH4 domain peptide. E11.5 embryonic kidneys were grown on filters for 20 h in medium containing either Chariot alone (control) or Chariot with the 10 M His 6 -tagged bcl-2 BH4 peptide. Prepared protein lysates were incubated with nickelagarose. The bound protein(s) were analyzed by Western blot analysis for paxillin. bcl-2, Paxillin, and Ureteric Bud Branching