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J. Biol. Chem., Vol. 276, Issue 37, 34567-34572, September 14, 2001

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The Development-associated Cleavage of Lens Connexin 45.6 by Caspase-3-like Protease Is Regulated by Casein Kinase II-mediated Phosphorylation^*

Xinye Yin, Sumin Gu, and Jean X. Jiang

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Received for publication, June 29, 2001, and in revised form, July 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu³⁶⁷ and Gly³⁶⁸. We have shown previously that Ser³⁶³ is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser³⁶³ was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The vertebrate lens is one of the most important model systems used in the study of the function and regulation of gap junctions. The lens is an avascular organ composed of an anterior epithelial cell layer and highly differentiated fibers ranging from the outer cortex toward the central core region. The terminal differentiation and aging of lens fibers are marked by dramatic morphological changes. As new cells arise on the outside of the lens, older cells are pushed inward where nuclei and organelles are lost (1). Because these cells are never lost from the lens, the most central cells are as old as the organism itself. The survival of lens cells relies on the intercellular communications between these cells and the cells at the lens surface through a large network of gap junctions that facilitate the exchange of ions and metabolites throughout the organ (2, 3).

Gap junctions are intercellular channels between two adjacent cells, which allow passage of small molecules (1000 Da) such as small metabolites, ions, and second messengers. The structural components of gap junctions are the members of a protein family called connexins, which consist of four conserved transmembrane domains and two extracellular loops, whereas their cytoplasmic regions are unique. The COOH terminus, the most variable region among connexins, contains several kinase and protease consensus sequences. Connexins are highly dynamic proteins that undergo rapid turnover both in cell lines (4) and in animal organs (5-7). The degradation has been identified to undergo either through the lysosome or proteasome pathway (8-12). Phosphorylation has been demonstrated to play an important role in regulation of gap junction stability and turnover (13-16). We have recently shown that an in vivo phosphorylated lens connexin undergoes a faster turnover than its unphosphorylated counterpart (17).

Differentiating lens fibers share a number of morphological and biochemical characteristics with cells undergoing apoptosis such as nucleus degeneration, loss of organelles, and activation of members of a cysteine protease family named caspase (18-21). However, unlike apoptotic cells, which are rapidly digested, the organelle-free lens fibers retain their basic cell integrity and metabolism throughout the lifetime of the organ. Poly(ADP-ribose) polymerase, an enzyme involved in DNA repair and maintenance of genomic integrity, is cleaved by caspases in the developing lens (18). Caspase-3 is predominantly activated during staurosporine-induced apoptosis in lens cells (22). The function of caspases has been reported to be responsible for nucleus degradation during the later stage of lens fiber differentiation (18). Although activation of caspases has been demonstrated during lens fiber differentiation, only a couple of caspase substrates have been identified, such as cytoskeletal proteins named - and -spectrins (21).

Connexin (Cx)¹ 45.6 is one of the connexins expressed in chick lens fibers (23, 24). Cx45.6, which is different from the other two chick lens connexins, Cx43 and Cx56, seems to be involved in lens development and differentiation. Mutations in Cx50, the mammalian ortholog of Cx45.6, have been identified in human cataract families (25, 26). Mice deficient in Cx50 not only develop cataracts, but also exhibit microphthalmia with a decrease of lens size (27). Primary cultures of chick lens closely mimic in vivo lens cell differentiation processes in which the monolayer of the lens epithelial cells differentiate into structures called "lentoids" displaying features of differentiated lens fibers (28, 29). We have recently shown that overexpression of Cx45.6 in lens primary cultures stimulates lens cell differentiation and formation of fibers (30). Additionally, Cx45.6 is subjected to post-translational modification in vivo. Cx45.6 is a phosphoprotein (23, 31). We have recently shown that Cx45.6 is phosphorylated in vivo by CKII at Ser³⁶³ located at the COOH-terminal region, and this phosphorylation facilitated the turnover of Cx45.6 (17). A previous report shows that Cx50, the ovine ortholog of Cx45.6, is also truncated at the COOH terminus in the center of the lens by a protease called calpain (32).

Although post-translational modifications, such as phosphorylation and specific truncation of lens connexins, have been investigated previously, the physiological significance and regulatory mechanism of these modifications in lens differentiation and development are less certain. In this report, we identified a specific proteolytic cleavage of Cx45.6 associated with lens development. Our experimental evidence suggests that the protease involved in this cleavage is caspase-3, an apoptotic protease. Moreover, the cleavage site by caspase-3 in Cx45.6 was identified to be close to the phosphorylated site by CKII. This specific phosphorylation protects Cx45.6 from cleavage by caspase-3. This is the first report showing that the apoptotic protease, caspase-3, is responsible for cleavage of a connexin, and this specific cleavage is regulated by a specific phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Fertilized chicken eggs were obtained from SPAFAS (Roanoke, IL) and Tyson Hatchery (Gonzalez, TX). CKII and caspase-3 were obtained from Sigma. Caspase-3 inhibitor Z-DEVD-FMK was from Calbiochem. [-³²P]ATP and [³⁵S]methionine were from PerkinElmer Life Sciences. Tissue culture reagents were purchased from Life Technologies, Inc. SDS-PAGE standards were from Bio-Rad. QuikChange^TM Site-directed Mutagenesis kit was obtained from Stratagene (La Jolla, CA). Chemiluminescence kit, ECL, was from Amersham Pharmacia Biotech. X-Omat AR film was obtained from Eastman Kodak.

Preparation of GST Fusion Proteins and Immunoaffinity Purification of Anti-Cx45.6 Antibody-- Bacterial fusion proteins containing GST fused with various portions of Cx45.6 were prepared as described (23, 33). The fusion proteins generated included the following: GST fused with partial COOH terminus of Cx45.6, GST-Cx45.6F1 (amino acids 307-389) and its corresponding site mutants, GST-Cx45.6F1(D364A) and GST-Cx45.6F1(E367A); GST fused with entire COOH terminus of Cx45.6, GST-Cx45.6F2 (amino acids 237-400); and GST fused with intracellular loop region of Cx45.6, GST-Cx45.6(L) (amino acids 98-148). Briefly, DNA fragments containing various portions of Cx45.6 or its mutants were produced by PCR with specific oligonucleotide primers. The codon GAT encoding Asp³⁶⁴ was changed to GCT encoding Ala and GAA encoding Glu³⁶⁷ was altered to GCA encoding Ala in one of the two primer sequences required for mutant synthesis. Each DNA fragment generated was inserted into the expression vector pGEX-2T. The recombinant fusion proteins were expressed in Escherichia coli, induced by isopropyl-thio--D-galactoside, and isolated with GST beads. To ensure the correct sequence all constructs generated were sequenced at the Institutional DNA Sequencing Facility. Fusion protein containing GST plus intracellular loop portion of Cx45.6 was used to raise polyclonal antisera in rabbits (Pocono Rabbit Farm and Laboratory Inc, Canadensis, PA). The antisera generated were immunoaffinity-purified by passage through two Sepharose CL-4B columns, GST-conjugated and GST-Cx45.6 fusion protein-conjugated, respectively, as described (23, 33).

SDS-PAGE and Western Blotting-- The fusion proteins and crude membrane samples were loaded in each lane of a 10% SDS-PAGE. For Western blotting, samples on SDS-PAGE were transferred to nitrocellulose membranes according to the method of White et al. (34). Membranes were probed with the affinity-purified preimmune or Cx45.6 antibody (1:500 dilution). The primary antibodies were detected either with alkaline phosphatase-conjugated secondary anti-rabbit antiserum or peroxidase-conjugated secondary anti-rabbit antiserum and followed by chemiluminescence reagent kit (ECL) (Amersham Pharmacia Biotech) according to the manufacturer's instruction. The membranes were exposed to X-Omat AR films (Eastman Kodak) and detected by fluorography.

Preparation of Retroviral Constructs and High Titer Retroviruses Containing Wild Type and Mutant Cx45.6-- Retroviral constructs and high titer viruses were prepared based on our protocol described previously (35). Briefly, cDNA fragment containing the wild type Cx45.6 was made by PCR and was constructed into the retroviral vector RCAS(A) as described (17, 36). With the wild type RCAS(A)-Cx45.6 DNA construct as a template, retroviral constructs of Cx45.6 mutants containing point mutations were generated with the QuikChange^TM Site-directed Mutagenesis kit according to the manufacturer's instructions. The codon alteration for site mutants, RCAS(A)-Cx45.6(D364A) and RCAS(A)-Cx45.6(E367A), were described above. RCAS(A)-Cx45.6(E367D) was made by changing codon GAA encoding Glu³⁶⁷ to GAT encoding Asp. All constructs generated were sequenced at the Institutional DNA Sequencing Facility. The high titer retroviruses (1-5 × 10⁸ colony forming units/ml) containing the wild type and the mutant RCAS(A) Cx45.6 constructs were prepared as we have described previously (35, 36).

Isolation of Cell Membranes from Lens or CEF Cells and in Vitro Cleavage by Caspase-3-- The crude cell membranes were isolated and prepared as described (37). Briefly, lenses from embryonic day 9 or CEF cells infected with retroviruses were lysed in lysis buffer (5 mM Tris, pH 8.0, and 5 mM EDTA/EGTA) plus 2 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 100 µM leupeptin. Crude membranes were pelleted at 35,000 rpm for 30 min (Beckman SW60 rotor).

The crude membranes from CEF cells and lenses or the purified GST fusion proteins were subjected to in vitro cleavage by caspase-3. Crude membranes or 2.5 µg of various GST fusion proteins were incubated at 37 °C in 25 µl of caspase-3 reaction buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM sucrose, and 5% glycerol) with and without 0.3 µg of caspase-3 at various times. Reactions were terminated by the addition of electrophoresis sample buffer (50 mM Tris, pH 6.8, 1% SDS, 2% -mercaptoethanol, and 35% glycerol) and boiled for 5 min.

Preparation of Primary Lens Cultures, Treatment with Apoptosis-inducing Reagents, and Inhibition of Caspase-3 Activity-- Primary lens cultures were prepared according to the procedure described previously (17, 38). Briefly, chick lenses dissected from embryonic day 11 embryos were ruptured, and the cells that dissociated from the lenses were plated at 1 × 10⁶ cells per 35-mm tissue culture plates in medium 199 plus 10% fetal bovine serum. Eight days after cell plating, primary cultures were treated either with 5 µM staurosporine dissolved in culture medium or with 2 mM EGTA for 4.5 h at 37 °C. When caspase-3 inhibitor Z-DEVD-FMK was used, cultures were preincubated with this inhibitor for 1 h before exposure to staurosporine or EGTA.

Culture of CEF Cells, Retroviral Expression, and Treatment with Apoptosis-inducing Reagents-- CEF cells were plated at 1 × 10⁵ cells in 35-mm tissue culture plates with DMEM plus 10% fetal calf serum and 5% CO₂. The 2nd day after cell plating CEF cells were infected with high titer retroviruses (5 µl/dish) carrying either the wild type or the mutant Cx45.6 cDNAs. When CEF cells reached confluence, they were collected either for membrane preparation or for treatment with apoptosis-inducing reagents. CEF cells were treated in the presence of 1 mM staurosporine for 8 h at 37 °C.

Phosphorylation of Cx45.6 by CKII, Radioactive Labeling of Lens Organ, in Vitro Cleavage by Caspase-3, and Immunoprecipitation-- The isolated embryonic lens membranes were incubated in 20 µl of CKII reaction buffer (10 mM MgCl₂, 50 mM MOPS, pH 7.0, 150 mM NaCl, and 20 µCi of [-³²P]ATP) plus 2 µl of CKII (119 units/ml) at 30 °C for 2 h. The reaction was stopped by the inactivation of CKII by incubating at 56 °C for 30 min. Fifty microliters of caspase-3 reaction buffer were added to the in vitro phosphorylated lens membrane sample, and the reaction mixture was then divided into two tubes. In each tube, 3 µl of caspase-3 or caspase-3 storage buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% CHAPS, and 10% (w/v) glycerol) was added and incubated at 37 °C for 6 h. The reaction was terminated by boiling the samples for 5 min in the presence of 0.6% SDS prior to the immunoprecipitation with affinity purified Cx45.6 antibody as previously described (31, 37). To control for the immunoprecipitation assay, embryonic day 9 lenses were rinsed three times with culture medium deficient in methionine prior to the incubation with 1 ml of ³⁵S-labeled medium (methionine-free DMEM with 5% dialyzed fetal calf serum, 20 µM methionine, and 0.5 mCi of [³⁵S]methionine) for 3 h. The radioactive medium was removed, and lenses were incubated in non-radioactive medium (DMEM supplemented with 0.5 mM methionine and 10% fetal calf serum) for various chase periods. ³⁵S-Labeled lens membranes were isolated and treated at the identical condition as the ³²P-labeled sample except the omission of ATP in CKII reaction mix, followed by the treatment by caspase-3, and immunoprecipitation as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Development-associated Cleavage of Lens Cx45.6-- Chick lenses at embryonic (E) days 8, 12, 15, and 18 and postnatal (P) days 1, 15, 30, and 60 were immunoblotted with affinity-purified Cx45.6 antibody that recognized the intracellular loop region of Cx45.6. The full-length Cx45.6 was phosphorylated in vivo and migrated as three close bands at the positions of 53, 56, and 58 kDa (Fig. 1) as demonstrated previously (23). At E8, a full-length Cx45.6 was solely present in the lens (Fig. 1, lane 1). Starting at E12, an additional band appeared migrating at around 46 kDa (empty arrowhead) (Fig. 1, lane 2) and its concentration level increased during the development of the lens as shown at E15 (Fig. 1, lane 3), E18 (Fig. 1, lane 4), and postnatal stages (Fig. 1, lanes 5-8). In addition, a 48-kDa fragment (solid arrowhead) closely migrating with the 46-kDa fragment appeared at E15, which is a lesser amount than 46-kDa fragment at embryonic stages. The level of this fragment, however, increased dramatically at the later developmental stages and reached a similar level as the 46-kDa fragment approaching P15 (Fig. 1, lane 6). No protein bands could be detected using pre-immune antibody (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Specific truncations of Cx45.6 were associated with lens development. Chick lenses were isolated at E day 8 (lane 1), 12 (lane 2), 15 (lane 3), and 18 (lane 4) and P day 1 (lane 5), 15 (lane 6), 30 (lane 7), and 60 (lane 8) and immunoblotted with affinity-purified Cx45.6 antibody, which recognizes the intracellular loop region of Cx45.6. The truncated Cx45.6 fragments are indicated by arrowheads.

To exclude the possibility that these cleavages may have occurred during sample preparation, we incubated lysates from embryonic and postnatal lenses at room temperature for 16 h and did not detect any accumulation of these fragments (data not shown). To determine if one of the closely migrated fragments was the phosphorylated form of another fragment, crude membranes from lens at P30 were treated with alkaline phosphatase. The levels of these two fragments were not altered (data not show), suggesting that neither of the fragments is the phosphorylated form of the other.

The Majority of the Cleaved Fragments Was Identified at the Central Core Region of the Postnatal Lenses-- The exterior portions of the 2-month postnatal chick lens containing epithelial cells and the fibers of the outer cortex were separated from the central core portion of the lens composed of mature lens fibers. The whole lens lysate contained both the full-length Cx45.6 and the cleaved fragments (Fig. 2, lane 3, arrowheads). However, the cleaved fragments predominantly existed in the mature fibers of the central core (Fig. 2, lane 1, arrowheads) and to a much lesser extent in the outer cortex which, in contrast, was mainly composed of full-length Cx45.6 (Fig. 2, lane 2). Because cells are retained in the lens and new cells continually arise on the outside of the lens, most cells in the central core regions are differentiated mature lens fibers (1). Consistent with the observation in Fig. 1 that truncated Cx45.6 fragments were accumulated associated with lens development, the results indicate that the specific cleavage of Cx45.6 in differentiated lens fibers may be related to the process of lens cell differentiation and formation of mature fibers.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   The majority of the truncated fragments was identified in the center of postnatal day 60 lenses. The exterior portions of the lens containing epithelial cells and outer fibers were separated from the central core region containing mature fibers. Cx45.6 expression in central core (lane 1), epithelia and outer cortex regions (lane 2), and entire lens lysate (lane 3) was examined by Western blots. The truncated fragments are shown as arrowheads.

The Development-associated Cx45.6 Cleavage Was Reproduced in Vitro by Caspase-3-- To identify the potential protease(s) involved in the cleavage of Cx45.6 during lens development, we first experimented with Ca²⁺-activated, cysteine protease calpain. This protease cleaves at the COOH terminus of Cx50, the ovine counterpart of Cx45.6 in the central region of the lens (32). Western blot of the membranes from embryonic (E) day 9 chick lens revealed a full-length Cx45.6 (Fig. 3A, lane 2). Treatment by calpain led to a cleavage from a full-length Cx45.6 into a major 33-kDa fragment (Fig. 3A, lane 3, open arrowhead), different from the 46-kDa fragment observed in the 2-month P chick lens (Fig. 3A, lane 1, solid arrowhead). This result suggests that unlike ovine Cx50 calpain is unlikely involved in the in vivo truncation of Cx45.6 in chick lens.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   In vitro cleavage of Cx45.6 by caspase-3. A, in vitro cleavage of Cx45.6 by calpain. Embryonic day 9 lens membranes were treated in the presence (lane 3) and absence (lane 2) of calpain at 37 °C for 2 h. The immunoblot with anti-Cx45.6 antibody shows that calpain cleaved the full-length Cx45.6 into a 33-kDa fragment (lane 3, indicated by empty arrowhead). This cleavage was not observed in the 2-month postnatal chick lens (lane 1), in which the 46-kDa fragments are indicated (solid arrowhead). B, cleavage of Cx45.6 by caspase-3 in lens membranes. Embryonic lens membranes were incubated with and without caspase-3 for 2 (lanes 1 and 2) and 16 h (lanes 3 and 4) and compared with Cx45.6 in newborn chick lens membranes (lane 5). The fragments generated by caspase-3 treatment migrated around 46 kDa, identical to the in vivo cleaved fragments from newborn lenses (arrowhead). C, cleavage of GST fusion proteins containing portions of Cx45.6 by caspase-3. GST protein (lanes 1 and 2) and GST fusion proteins containing the intercellular loop (GST-45.6L) (lanes 3 and 4), partial COOH terminus (GST-45.6F1, amino acids 307-389) (lanes 5 and 6), or entire COOH terminus (GST-45.6F2, amino acids 237-400) (lanes 7 and 8) were treated in the presence (lanes 2, 4, 6, and 8) and absence (lanes 1, 3, 5, and 7) of caspase-3 at 37 °C for 6 h. The cleaved fragments are indicated by arrowheads.

Caspase-3-like protease involved in apoptosis has been reported to be activated when lens epithelial cells differentiate into fibers in vivo (39), and caspase-3 expression is localized in the outer fibers (18). Caspase-3 is activated between embryonic days 8 and 12 in chick lens (18), which is at the similar time frame as when the cleavage of Cx45.6 was observed. To determine whether caspase-3 is the protease responsible for the cleavage, embryonic lens membranes were incubated with caspase-3. As shown in Fig. 3B, after 2-h incubation, the full-length Cx45.6 was partially cleaved into fragments around 46 kDa (Fig. 3B, lane 1, arrowhead), which migrated to the same position as the truncated Cx45.6 of the lens membrane from the newborn chick (Fig. 3B, lane 5, arrowhead). After 16-h incubation with caspase-3, the majority of the full-length Cx45.6 proteins was cleaved (Fig. 3B, lane 3, arrowhead).

These results indicate that Cx45.6 may be a direct substrate for caspase-3. Alternatively, Cx45.6 can be cleaved by other protease(s) present in the lens membrane that could be activated by caspase-3. To examine these possibilities further, purified fusion proteins containing GST fused with portions of Cx45.6 were used for in vitro caspase-3 enzymatic digestion. The fusion parts of Cx45.6 include the following: the intracellular loop of Cx45.6 (GST-45.6L) (amino acids 98-148) (Fig. 3C, lanes 3 and 4), partial COOH terminus (GST-45.6 F1) (amino acids 307-389) (Fig. 3C, lanes 5 and 6), and entire COOH terminus (GST-45.6F2) (amino acids 237-400) (Fig. 3C, lanes 7 and 8). These fusion proteins, together with the GST protein itself (Fig. 3C, lanes 1 and 2), were treated with caspase-3. Both GST-45.6 F1 (Fig. 3C, lane 6) and GST-45.6F2 (Fig. 3C, lane 8) were cleaved by caspase-3 (arrowheads), whereas GST protein (Fig. 3C, lane 2) and GST-45.6L (Fig. 3C, lane 4) were not cleaved. These results suggest that Cx45.6 is a substrate of caspase-3 and that the cleavage site is located within the COOH terminus between amino acids 307 and 389.

Development-associated Truncation of Cx45.6 Was Reproduced by a Caspase-3-like Protease Following the Treatment of Apoptosis-inducing Reagents in Lens Cells-- To examine whether Cx45.6 can be cleaved by caspase-3 in lens cells, primary lens cultures were incubated with apoptosis-inducing reagents staurosporine (Fig. 4, lane 2) (22) or EGTA (Fig. 4, lane 4) (40). These two compounds are known chemical inducers for caspase-mediated apoptosis (22, 40). Treatments by both reagents regenerated fragments around 46 kDa (Fig. 4, lanes 2 and 4, arrowhead). The cleavage was significantly inhibited by Z-DEVD-FMK, a caspase-3 inhibitor (Fig. 4, lane 5). The results confirm that caspase-3 is likely to be involved in the cleavage of Cx45.6 in vivo.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   The development-related Cx45.6 cleavage was reproduced following the treatment of apoptotic reagents in lens primary cultures. The lens primary cultures were treated in the presence (lane 2) and absence (lane 1) of 5 µM staurosporine (STS) and in the absence (lane 3) and presence (lane 4) of 2 mM EGTA for 4.5 h. One of the samples was pretreated with caspase-3 inhibitor Z-DEVD-FMK (lane 5) for 1 h prior to the treatment by EGTA. The cleaved fragments are indicated (an arrowhead).

Caspase-3 Cleaved Cx45.6 between Amino Acid Residues Glu³⁶⁷ and Gly³⁶⁸-- The amino acid sequence Asp³⁶⁴-Glu-Val-Glu³⁶⁷-Gly was identified in the COOH-terminal region of Cx45.6 (see Fig. 5A). This sequence is similar to a known caspase-3 consensus sequence (41) with the exception of substituting Asp with Glu at position 367. If cleavage by caspase-3 occurs between Glu³⁶⁷ and Gly³⁶⁸, the predicted molecular weight of one of the cleaved fragments would match that of the 46-kDa fragment. The two aspartate residues in the consensus sequence of caspase-3 are essential, and cleavage cannot occur if either residue is altered to alanine (42). Since glutamate like aspartate is an acidic residue, this segment of the sequence in Cx45.6 is a potential target for caspase-3. As illustrated in Fig. 5A, mutants were constructed with alterations in the conserved amino acid residues corresponding to the consensus sequence of caspase-3. Fusion proteins GST-Cx45.6F1, and its mutants GST-Cx45.6F1(D364A) and GST-Cx45.6F1(E367A), were subjected to caspase-3 cleavage in vitro (Fig. 5B). GST-Cx45.6F1 was cleaved by caspase-3 into two closely migrating bands (Fig. 5B, lane 2). Both GST-Cx45.6F1(D364A) (Fig. 5B, lane 4) and GST-Cx45.6F1(E367A) (Fig. 5B, lane 6) exhibited resistance to cleavage by caspase-3. These data suggest that either Asp³⁶⁴ or Glu³⁶⁷ seems to be crucial in the cleavage by caspase-3.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Caspase-3 cleaved Cx45.6 between amino acid residues Glu367 and Gly368. A, caspase-3 consensus sequence and its corresponding sequences in Cx45.6 of wild type (WT) and site mutants, Cx45.6(D364A), Cx45.6(E367A), and Cx45.6(E367D), are illustrated. The cleavage site is marked by an arrow (B). Fusion protein GST-45.6F1 (lanes 1 and 2) and its mutants, GST-45.6F1(D364A) (lanes 3 and 4) and GST-45.6F1(E367A) (lanes 5 and 6), were treated with (lanes 2, 4, and 6) and without (lanes 1, 3, and 5) caspase-3. The cleaved fragments are indicated by an arrowhead. C, exogenous full-length Cx45.6 (lanes 1 and 2) and its corresponding site mutants, Cx45.6(D364A) (lanes 3 and 4), Cx45.6(E367A) (lanes 5 and 6), and Cx45.6(E367D) (lanes 7 and 8), were expressed in CEF cells through retroviral RCAS(A) infection. Crude membranes of Cx45.6 expressed in CEF cells were treated with (lanes 2, 4, 6, and 8) and without (lanes 1, 3, 5, and 7) caspase-3, and the fragments resulting from digestion reaction are indicated by an arrowhead. D, CEF cells expressing wild type and mutant Cx45.6 through retroviral RCAS(A) infection were treated with (lanes 2, 4, 6, and 8) and without (lanes 1, 3, 5, and 7) 1 mM staurosporine (STS) for 8 h. The cleaved fragments are indicated (arrowhead).

The COOH terminus of Cx45.6 in GST fusion protein may not have similar conformation as compared with the one in the membrane-spanning full-length molecule. Therefore, full-length Cx45.6 and its mutants carrying alterations of conserved residues corresponding to caspase-3 (see Fig. 5A) were expressed in CEF cells through retroviral infection, and the isolated cell membranes were subjected to caspase-3 treatment (Fig. 5C). Similar to the results shown for fusion proteins, mutations in D364A and E367A prevented cleavage of Cx45.6 by caspase-3 (Fig. 5C, lanes 4 and 6). Furthermore, the mutation of E367D, producing a cleavage sequence in Cx45.6 identical to the consensus sequence for caspase-3, increased cleavage (Fig. 5C, lane 8). The ratio of the fragments around 46 kDa to the full-length form of Cx45.6(E367D) (Fig. 5C, lane 8) is greater than that to the full-length wild type Cx45.6 (Fig. 5C, lane 2).

Similar results were observed when CEF cells expressing wild type and mutant Cx45.6 were treated by the apoptosis-inducing reagent staurosporine (Fig. 5D). Neither Cx45.6(D364A) nor Cx45.6(E367A) was cleaved (Fig. 5D, lanes 4 and 6), whereas the wild type Cx45.6 was (Fig. 5D, lane 2). In comparison to the wild type Cx45.6, the cleavage of Cx45.6(E367D) was further enhanced (Fig. 5D, lane 8). Together, the results obtained from fusion proteins and full-length Cx45.6 expressed in cells suggest that the cleavage site of Cx45.6 by caspase-3 is between Glu³⁶⁷ and Gly³⁶⁸.

Inhibition of Caspase-3-catalyzed Cleavage of Cx45.6 by Phosphorylation of Cx45.6 by CKII-- We have reported that Ser³⁶³, an amino acid residue close to the caspase-3 cleavage site, is phosphorylated in vivo by CKII (17). Phosphorylation of certain substrates has been shown to be important in regulating their cleavage by caspase-3 (42, 43). To determine whether phosphorylation at Ser³⁶³ affects the ability of Cx45.6 to serve as a substrate for caspase-3, Cx45.6 in the membranes of embryonic lenses was treated in the presence of CKII and followed by proteolytic digestion by caspase-3 (Fig. 6, lane 2). The results indicate that Cx45.6 phosphorylated by CKII was protected from caspase-3 digestion. To ensure that Cx45.6 in both full-length and truncated forms could be immunoprecipitated, embryonic lenses were metabolically labeled with [³⁵S]methionine and chased in non-radioactive medium immediately (Fig. 6, lanes 3 and 4) and after 6 h of incubation (Fig. 6, lanes 5 and 6). The isolated membranes labeled with [³⁵S]methionine were treated in the presence (Fig. 6, lanes 4 and 6) and absence (Fig. 6, lanes 3 and 5) of caspase-3 and followed by immunoprecipitation. The Cx45.6 antibody we used here was capable of immunoprecipitating the full-length as well as the truncated (arrowhead) Cx45.6 cleaved by caspase-3. Together, the data suggest that the specific cleavage of Cx45.6 by caspase-3 is likely to be regulated by CKII-mediated phosphorylation.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Phosphorylation of Cx45.6 by CKII inhibited its cleavage by caspase-3. Crude membranes of embryonic lenses were phosphorylated by CKII and [-32P]ATP and followed by incubation in the presence (lane 2) and absence (lane 1) of caspase-3. Cx45.6 was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The control experiment was conducted with embryonic lenses labeled with [35S]methionine for 3 h and chased in non-radioactive medium for 0 (lanes 3 and 4) and 6 h (lanes 5 and 6). The crude membranes from [35S]methionine-labeled lenses were incubated in CKII reaction mix without ATP followed by the treatment with (lanes 4 and 6) and without (lanes 3 and 5) caspase-3 and immunoprecipitation by Cx45.6 antibody. The cleaved fragments are indicated by an arrowhead.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we identified caspase-3 as the protease responsible for the cleavage of Cx45.6 during chick lens development. Additionally, the cleavage on Cx45.6 was localized to the site close to the amino acid residue phosphorylated by CKII. Our previous studies (17) have shown that Cx45.6 is phosphorylated in vivo by CKII at Ser³⁶³, and this phosphorylation may be a signal for Cx45.6 degradation mediated by proteasome. Here, we found that phosphorylation of Ser³⁶³ by CKII protected Cx45.6 from cleavage by caspase-3, suggesting that the specific cleavage of Cx45.6 is regulated by CKII-mediated phosphorylation.

Based on our experimental evidence, a model is proposed for the involvement of CKII-mediated phosphorylation in the regulation of Cx45.6 turnover and specific cleavage by caspase-3-like protease. In the outer cortex of the lens, when a full-length Cx45.6 is phosphorylated by CKII at Ser³⁶³, it becomes unstable and possibly undergoes degradation mediated through the proteasome pathway (17). The CKII phosphorylation site Ser³⁶³ is located within a PEST domain (17, 44), and this domain has been reported to be associated with a rapid protein turnover via proteasome-mediated degradation (45). Additionally, phosphorylation is also known to signal protein degradation by proteasome (46). When Ser³⁶³ is not phosphorylated by CKII, Cx45.6 renders itself into a substrate for caspase-3, which cleaves at Glu³⁶⁷ at the COOH terminus of Cx45.6 and generates truncated fragments of Cx45.6. These fragments were localized to the central core region of the lens.

We observed two fragments derived from Cx45.6 in the size of 46 and 48 kDa. These fragments were accumulated during lens development. The 46-kDa fragment was predominant during the embryonic stage, whereas the level of the 48-kDa fragment increased and reached a similar level as compared with the 46-kDa form at the postnatal stage. Formation of two fragments was also observed when GST-Cx45.6F1 was treated with caspase-3. However, mutation of a potential cleavage site in GST-Cx45.6F1 blocked the production of either fragment, suggesting that this site initiates generation of these two fragments. Because of the closeness of their migration, these two fragments sometimes were less distinctive on the SDS-PAGE. We also observed a potential third band larger than 48 kDa at P60 samples, and this band could be generated from additional cleavage or certain type of posttranslational modifications.

Our data suggest that caspase-3-like protease is responsible for the in vivo cleavage of Cx45.6. In contrast to our observation in chicken, a previous study has shown that a large portion of the COOH terminus of Cx50, the ovine ortholog of Cx45.6, is removed by calpain (32). This discrepancy could account for the difference in expression level or accessibility of these two proteases expressed in these two species, because the responsibility of one protease could be replaced by the other during evolution. It is also likely that the cleaved fragment of Cx50 observed in ovine is caused by in vitro experimental manipulation. We found if lysates of lens primary cells were handled improperly, Cx45.6 would be cleaved into a 33-kDa fragment, an identical size fragment as the fragment cleaved by calpain (data not shown).

We have also shown that the truncated Cx45.6 was accumulated during lens development and was localized to the differentiated lens fibers. The activation of caspases-3 also initiates in the differentiated lens fibers and is involved in later stage cell differentiation, such as nucleus degeneration (18). Thus, the caspase-3-truncated Cx45.6 is likely to form a unique type of gap junctions, different from the ones formed by full-length Cx45.6. These intercellular channels may facilitate the passage of factors important for fiber differentiation and/or maintain the physiological functions of mature fibers. Functional gap junction channels are, indeed, formed by truncated Cx50 of ovine lens (32). These channels are characterized to be less sensitive to low pH, compared with those formed by full-length Cx50. Studies by Le and Musil (47) have shown that lens primary cultures differentiate normally even if the channels are blocked with the gap junction inhibitor 18-glycyrrhetinic acid. Since 18-glycyrrhetinic acid has been tested for gap junctions formed solely by full-length connexins (48), the channels consisting of truncated connexins may not be similarly inhibited by this chemical.

Lens Cx45.6 is the first connexin identified as a substrate for caspase. In addition to lens fibers other cell types, such as skin keratinocytes, also undergo degeneration of nuclei and organelles during cell differentiation. Caspase activation has been identified in the differentiation of epidermal keratinocytes and is required for the loss of the nucleus (49). It is of great interest to determine whether connexins in those cells are also subjected to regulation by caspases. The physiological significance of the regulation of connexins by caspases requires further investigation.

	ACKNOWLEDGEMENTS

We thank D. Adan-Rice and L. Wang for technical assistance and Z. Dong for valuable discussions. We thank members of the Jiang laboratory for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant EY-12085 (to J. X. J.).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.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-3796; Fax: 210-567-6595; E-mail: jiangj@uthscsa.edu.

Published, JBC Papers in Press, July 11, 2001, DOI 10.1074/jbc.M106073200

	ABBREVIATIONS

The abbreviations used are: Cx, connexin; CKII, casein kinase II; CEF, chicken embryonic fibroblast; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E, embryonic day; P, postnatal day.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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