©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conversion from Oligomers to Tetramers Enhances Autophosphorylation by Lens A-Crystallin
SPECIFICITY BETWEEN A- AND B-CRYSTALLIN SUBUNITS (*)

(Received for publication, December 23, 1994)

Marc Kantorow (1), Joseph Horwitz (2), Martinus A. M. van Boekel (3), Wilfried W. de Jong (3), Joram Piatigorsky (1)(§)

From the  (1)Laboratory of Molecular and Developmental Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, the (2)Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90024, and the (3)Department of Biochemistry, University of Nijmegen, 6525 EK Nijmegen, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously we showed that -crystallins are autophosphorylated (Kantorow, M., and Piatigorsky, J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3112-3116). Here we report that addition of 1% deoxycholate converted A-crystallin aggregates into 80-kDa tetramers which were 10-fold more active for autophosphorylation. Circular dichroism (CD) spectra of -crystallin revealed little or no change in secondary and tertiary structures in 1% deoxycholate. A2D, a truncated form of bovine A that exists as a tetramer, was as active for autophosphorylation in the absence of deoxycholate as intact A was in the presence of deoxycholate. At least one serine between amino acids 131 and 145 of bovine A was autophosphorylated in peptide mapping experiments. Chicken A-crystallin, which lacks the Ser-122 cAMP-dependent kinase site of bovine A, was also autophosphorylated in the presence of deoxycholate. In contrast to A-crystallin, autophosphorylation by B-crystallin was not activated by deoxycholate despite its conversion to a tetrameric form, and B was also more efficiently phosphorylated by cAMP-dependent kinase than A. These data suggest metabolic differences between the -crystallin subunits that may be related to specific expression of A in the lens and ubiquitous expression of B in numerous normal and diseased tissues.


INTRODUCTION

The two -crystallins (A and B) are major proteins expressed in all vertebrate eye lenses where they contribute to lens transparency(1, 2) . A (3, 4) and especially B (5, 6) are also expressed constitutively in other tissues. In addition to its expression in normal tissues, B-crystallin has been associated with changes in cellular morphology during embryogenesis(7, 8) , with neurodegenerative diseases(9, 10, 11, 12, 13, 14, 15) , with fibroblasts from patients with Werner's disease(16) , with harmatomas(17) , and with neuroectodermal tumors (18, see (19) for additional references).

A and B belong to the family of small heat shock proteins (shsps) (20, see (21) for additional references) and can form complexes with other shsps(22, 23, 24) . B is inducible by heat and other physiological stress(25, 26) . A and B are also molecular chaperones that associate with and can prevent denaturation of other proteins(27, 28) .

The 173-amino-acid A- and 175-amino-acid B-crystallin polypeptides undergo a large variety of post-translational modifications(29) . One of these is serine-specific phosphorylation which occurs via a cAMP-dependent pathway in crude lens extracts(30, 31, 32, 33) . In vivo phosphorylation of bovine A-crystallin occurs predominately on serine 122(32, 33) , but at least three in vivo phosphorylation sites in addition to Ser-122 have been identified between amino acids 122 and 173 in peptide mapping experiments(34) . In addition to Ser-122, a second phosphorylation site located between amino acids 35 and 57 has been reported for human A(35) . In vivo phosphorylation of bovine B-crystallin occurs on serines 19, 45, and 59(36, 37, 38) . An additional serine between amino acids 29 and 38 has also been reported (34) . Serines 122 and 148 in bovine A and 59 in bovine B are contained in a potential ((Arg/Lys)-(X)-Pro-Ser) cAMP-dependent kinase recognition site, while the other phosphorylated serines in bovine A and bovine B are not(34) .

Extracted -crystallin exists as a multisubunit complex with an average molecular mass of 800 kDa(39, 40) . We have recently demonstrated that purified -crystallin can be phosphorylated in the absence of cAMP by a serine-specific, Mg-dependent, autophosphorylation reaction (41) . Here we provide evidence that autophosphorylation of A- but not B-crystallin is activated by reducing the average molecular mass of the A complex from approximately 800 kDa to 80 kDa, and we provide evidence for functional differences between A- and B-crystallin subunits with respect to phosphorylation.


EXPERIMENTAL PROCEDURES

Preparation of -Crystallin Polypeptides

Bovine A and B were prepared from the outer cortex of bovine lenses as described elsewhere(27) . In brief, total -crystallin was purified by gel filtration on a Sephacryl S-200 column followed by FPLC()purification on a Superose 6HR 10/30 prepacked column; bovine A and B polypeptides were obtained using a Bio-Rad Rotofor preparative isoelectric focusing cell in the presence of 8 M urea. Recombinant A or A2D were expressed and purified as described(42) . Total chicken -crystallin was purified from 15-day-old embryonic lenses as described for bovine -crystallin (Horwitz, 1992).

Gel Filtration Chromatography

Gel filtration chromatography was performed in the presence of 1% deoxycholate on a 1.5 90 cm column packed with Pharmacia Sephacryl HR200. This was driven by a Pharmacia FPLC system. Samples of -crystallin (1-8 mg) were solubilized in 1% deoxycholate and applied directly to the column. The elution buffer was 1% deoxycholate, 20 mM Tris-HCl (pH 7.9), and 0.1 M NaCl. The flow rate was 0.5 ml/min, fractions of 2 ml were collected, and the absorbance at 280 nm was recorded. To calibrate the column, the following proteins were chromatographed in the presence of 1% deoxycholate: aldolase (158 kDa), enolase (88 kDa), bovine serum albumin (68 kDa), and -crystallin (20 kDa). Aldolase and enolase did not dissociate under these conditions.

CD Spectra of A-Crystallin

These were carried out using a Jasco model 600 spectropolarimeter. For the near and far UV regions, 1.0-cm and 0.20-mm path length cells were used, respectively.

In Vitro Phosphorylation

In vitro phosphorylation reactions were carried out as specified in 20 mM imidazole HCl (pH 7.4), 1 mM MgCl, [-P]ATP at 37 °C in the presence or absence of 1% deoxycholate, CHAPS, n-octyl glucopyranoside, or SDS. Where indicated, crystallins were reacted with bovine heart cAMP-dependent kinase (Sigma) under identical conditions with the addition of 20 µM cAMP. Reactions were terminated by the addition of 10% SDS buffer (10% (w/v) SDS, 0.5 M Tris-HCl (pH 6.8), 5% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol), loaded onto precast SDS, 14% or 4-20% polyacrylamide gels (Novex, San Diego, CA), and electrophoresed according to the recommendations of the manufacturer. After electrophoresis, the proteins were transferred (30 V for 2 h in 12 mM Tris-HCl, 96 mM glycine, 15% methanol) to nitrocellulose filters and stained with Ponceau S. Autoradiography was performed at -70 °C overnight on Kodak X-Omat AR film with an intensifying screen.

Reversed-phased HPLC Analysis of [P]A-Crystallin Tryptic Peptides

1-2 mg of recombinant A-crystallin were autophosphorylated as described above in the presence or absence of 1% deoxycholate or cAMP-dependent kinase with 5 µM [-P]ATP (4500Ci/mmol). Free ATP was removed by dialysis, and the resulting protein was aminoethylated and digested with trypsin. The resulting peptides were separated by reversed-phase HPLC and analyzed by scintillation counting as described(32) .


RESULTS

Deoxycholate Disaggregates the A- and B-Crystallin Multisubunit Complexes to 80-kDa Tetramers

The aggregation state of A- and B-crystallin subunits was determined by chromatography in the presence of 1% deoxycholate, CHAPS, n-octyl glucopyranoside, or SDS. Fig. 1is the profile of recombinant A-crystallin chromatographed in the presence of 1% deoxycholate. The majority of A-crystallin subunits eluted with a retention time corresponding to a molecular mass of 80 kDa, consistent with a tetramer. Aldolase tetramers (158 kDa) and enolase dimers (88 kDa) consistently ran in front of the A tetramer. Identical results were obtained with highly purified bovine A-crystallin and B-crystallin (data not shown). The presence of 1% CHAPS or 1% n-octyl glucopyranoside did not result in disaggregation of A- or B-crystallin subunits (data not shown). As expected, 1% SDS converted A- or B-subunits to 20-kDa monomers when analyzed by chromatography (data not shown).


Figure 1: Deoxycholate converts A-crystallin from a large molecular mass aggregate to a tetrameric form. Recombinant A-crystallin was analyzed by chromatography in the presence of 1% deoxycholate. Aldolase (158 kDa), enolase (88 kDa), bovine serum albumin (68 kDa), and -crystallin (20 kDa) were molecular mass standards. The majority of A-crystallin eluted with fraction 42 (84 min) corresponding with a molecular size of 80 kDa. Note that aldolase tetramers and enolase dimers elute before A tetramers.



Deoxycholate Does Not Alter the Secondary or Tertiary Structure of A-Crystallin

Fig. 2A shows the far UV circular dichroism (CD) spectra of recombinant A-crystallin in the presence of deoxycholate, SDS, and aqueous buffer. In deoxycholate (curve 2), there was a slight (10%) increase in the intensity of the spectrum (from 200-230) when compared to buffer alone (curve 1); however, no change in shape was observed. This typical CD spectrum for A-crystallin reflects its predominantly -structure. In contrast, major changes occurred when A was solubilized in 1% SDS (curve 3). This spectrum suggests a significant amount of -helical structure. SDS is known to induce helicity in nonhelical proteins. The near UV CD spectra of recombinant A-crystallin in deoxycholate, SDS, and aqueous buffer are shown in Fig. 2B. The vibronic structure in deoxycholate and aqueous buffer was essentially the same; by contrast, the vibronic structure in SDS due to tryptophan residues at 283 nm and at 292 nm is not present, reflecting modifications in the microenvironments of tryptophan and possibly tyrosine residues. Collectively, these CD spectra indicate minor changes in the secondary and tertiary structures of A-crystallin in 1% deoxycholate and major conformational changes upon monomerization in 1% SDS.


Figure 2: A, far UV CD spectra of A-crystallin in the presence or absence of deoxycholate and SDS. The samples were dissolved as described. Recombinant A-crystallin A = 1.5 was dissolved in either 20 mM Tris buffer, pH 7.9, and 0.1 M NaCl (curve 1), +1% deoxycholate (curve 2; broken line), and +1% SDS (curve 3). SDS buffer alone is shown as a solid line. The path length was 1 cm. Each curve represents the average of 16 scans. B, near UV CD spectra of A-crystallin in the presence or absence of deoxycholate and SDS. Recombinant A-crystallin A = 1.5 was dissolved either in 20 mM Tris buffer, pH 7.9 and 0.1 M NaCl (curve 1), same as above + 1% deoxycholate (curve 2), or same as above + 1% SDS (curve 3). The path length was 0.2 mm. Each spectrum represents the average of eight scans.



Autophosphorylation of A-Crystallin Is Enhanced by Tetramerization

The relative amounts of autophosphorylation of recombinant A in the presence or absence of 1% deoxycholate, CHAPS, n-octyl glucopyranoside, and SDS were estimated by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 3). Deoxycholate activated autophosphorylation by A (lanes 2 and 7) to levels 10 times those measured in the absence of this detergent (lanes 1 and 6). CHAPS (lanes 3 and 8) or n-octyl glucopyranoside (lanes 4 and 9), which do not disaggregate A-crystallin, had no effect on autophosphorylation. SDS (lanes 5 and 10), which converts A-crystallin to a monomer, promoted at most a 2-fold increase in autophosphorylation. Identical results were obtained with bovine A-crystallin purified by isoelectric focusing in the presence of 8 M urea (data not shown).


Figure 3: Deoxycholate increases autokinase activity of recombinant A-crystallin. A, recombinant A-crystallin (10 µg) was incubated in the absence (lanes 1 and 6) or presence of 1% deoxycholate (DOC) (lanes 2 and 7), CHAPS (lanes 3 and 8), n-octyl glucopyranoside (NOG) (lanes 4 and 9) or sodium dodecyl sulfate (SDS) (lanes 5 and 10). All reactions were performed in 20 mM imidazole, pH 7.4, 1 mM MgCl, and 50 µM [-P]ATP, specific activity 1.6 Ci/mmol for 1 h at 37 °C. Autophosphorylation was monitored by SDS-PAGE and autoradiography. Lanes 1-5 are the Ponceau S stained blot, and lanes 6-10 the corresponding autoradiograph. The positions of A-crystallin and molecular mass standards are indicated. Also indicated by an asterisk is a weak band identified by Western analysis to contain A-crystallin. Densitometric values for each sample are given relative to A + deoxycholate.



Specificity of Autophosphorylation in the Presence of 1% Deoxycholate

In order to test the possibility that the enhanced phosphorylation of A-crystallin in the presence of deoxycholate was due to a nonspecific chemical effect, eight different proteins were examined in parallel with recombinant A-crystallin for autophosphorylation (Fig. 4). While A-crystallin was intensely labeled, only three other protein preparations (carbonic anhydrase, ovalbumin, and lactate dehydrogenase) showed minor radioactivity on the polyacrylamide gel. The label in the carbonic anhydrase lane migrated considerably below the major band of carbonic anhydrase, while the label in ovalbumin and lactate dehydrogenase lanes migrated slightly above the major bands of these proteins. Similar results were obtained when the reactions were conducted in the absence of deoxycholate albeit with higher background radioactivity (data not shown). In our previous study(41) , high specific activities (up to 7500 Ci/mmol) of [-P]ATP were used to monitor -crystallin autophosphorylation. In the present tests, much lower specific activities (1-2 Ci/mmol) were used which decreased nonspecific labeling. We have repeated these experiments in the presence of A-crystallin ((+) or(-)-deoxycholate) and have found only specific labeling of A-crystallin, indicating that the phosphate group was not transferred from A to the other proteins examined (data not shown).


Figure 4: Specificity of autophosphorylation in the presence of deoxycholate. Recombinant A (10 µg, reA) (lanes 1 and 9), carbonic anhydrase (10 µg, CA) (lanes 2 and 10), chicken -crystallin (10 µg, chic) (lanes 3 and 11), ovalbumin (5 µg, OA) (lanes 4 and 12), trypsin inhibitor (5 µg, TI) (lanes 5 and 13), -casein (10 µg, Cas) (lanes 6 and 14), lactate dehydrogenase (5 µg, LDH) (lanes 7 and 15), and myelin basic protein (10 µg, MBP) (lanes 8 and 16) were reacted in 20 mM imidazole, pH 7.4, 1 mM MgCl, and 50 µM [-P]ATP, specific activity 1.6 Ci/mmol for 1 h at 37 °C. Autophosphorylation was monitored by SDS-PAGE and autoradiography. Lanes 1-8 are the Ponceau S-stained blot, and lanes 9-16 the corresponding autoradiograph. The positions of A-crystallin molecular mass standards are indicated.



Enhanced Autophosphorylation Activity by Truncated Recombinant A-Crystallin

Previous work demonstrated that deletion of amino acids 1-63 of bovine A-crystallin results in a tetrameric form of A called A2D that corresponds to the putative C-terminal domain of A(42) . Thus, it was hypothesized that tetrameric A2D would exhibit high levels of autophosphorylation in the absence of deoxycholate, provided that amino acids 1-63 are not required for autokinase activity.

Autophosphorylation of A2D in the absence of deoxycholate (Fig. 5, lanes 3, 4, 9, and 10) was at least five times that of untreated bovine A-crystallin (relative to mass) (lanes 6 and 12) and almost as high as that of bovine A-crystallin treated with deoxycholate (lanes 5 and 11). Unexpectedly, 1% deoxycholate (lanes 1, 2, 7, and 8) actually reduced the autophosphorylation of A2D by approximately 50%. The autophosphorylation of a form of A-crystallin (indicated in Fig. 3as A*), identified by Western analysis as a nonreducible dimer of A-crystallin(41) , was unaffected by deoxycholate (lanes 5, 6, 11, and 12). A2D also showed a more slowly migrating, nonreducible band by SDS-polyacrylamide electrophoresis (lanes 1-4 and 7-10). This band has been designated A2D* by analogy with A* and is presumed to be a dimer of A2D. In contrast to A*, deoxycholate significantly reduced the autophosphorylation of A2D* (compare lanes 7 and 8 with deoxycholate with lanes 9 and 10 without deoxycholate).


Figure 5: The effect of deoxycholate on autophosphorylation by A2D. A2D (10 µg) (lanes 1-4 and 7-10) and bovine A-crystallin (10 µg) (lanes 5, 6, 11, and 12) were incubated in the presence or absence of 1% deoxycholate in 20 mM imidazole, pH 7.4, 1 mM MgCl with 0.01 µM, 4500 Ci/mmol [-P]ATP for 45 min. Autophosphorylation was monitored by SDS-PAGE and autoradiography. Lanes 1-6 are the Ponceau S-stained blot and lanes 7-12 the corresponding autoradiograph. The positions of A2D, A, and molecular mass standards are indicated. Also indicated are A* and A2D*. Densitometric values for each sample are given relative to A + deoxycholate.



Autophosphorylation of Chicken A-Crystallin

The major in vivo phosphorylation site for bovine A-crystallin in response to exogenously added cAMP is serine 122, which is contained in the cAMP-dependent recognition site RLPS(33) . Unlike bovine A, chicken A is not phosphorylated by cAMP-dependent kinase (32) because it contains alanine instead of serine at 122. Thus, chicken A was examined for autokinase activity to determine if serines other than 122 could be involved in autophosphorylation. Chicken A-crystallin exhibited deoxycholate-activated autophosphorylation (Fig. 6, lanes 2 and 4). There was almost no autophosphorylation of the chicken A- or B-crystallin polypeptides in the absence of deoxycholate (lane 3). In contrast to its effect on A, deoxycholate did not stimulate autophosphorylation of B (lane 4). Since chicken A has alanine substituted for serine at position 122, its autophosphorylation must involve other amino acids.


Figure 6: Chicken A-crystallin is capable of deoxycholate-activated autophosphorylation. Total chicken -crystallin (5 µg) was incubated in the presence or absence of 1% deoxycholate in a reaction mixture containing 20 mM imidazole, pH 7.4, with 50 µM [-P]ATP, specific activity 1 Ci/mmol for 1 h at 37 °C. Autophosphorylation was monitored by SDS-PAGE and autoradiography. Lanes 1 and 2 are the Ponceau S-stained blot and lanes 3 and 4 the corresponding autoradiograph. The positions of chicken A- and B-crystallins and molecular mass markers are indicated.



Analysis of Autophosphorylated Bovine A-Crystallin by Reversed-phase HPLC Separation of Tryptic Peptides

Autophosphorylation of chicken A-crystallin eliminates serine 122 as an autophosphorylation site in that species and makes it unlikely that Ser-122 is autophosphorylated in the bovine polypeptide, as it is in the cAMP-dependent phosphorylation reaction(32) . In addition, no differences were observed in autophosphorylation of phosphorylated and unphosphorylated forms of A-crystallin purified from bovine lens by isoelectric focusing (data not shown). Consequently, we attempted to identify the autophosphorylated amino acids of bovine A-crystallin by reversed-phase HPLC separation of tryptic peptides. A control test on A phosphorylated by cAMP-dependent kinase confirmed the labeling of tryptic peptide T16-T17a (the result of incomplete cleavage between T16 and T17a, which always occurs), indicative of Ser-122 phosphorylation(32) . Three separate preparations of autophosphorylated bovine recombinant A-crystallin were examined. In the first, A was autophosphorylated in the absence of deoxycholate, aminoethylated, digested with trypsin, and subjected to HPLC chromatography. Only two major tryptic peptides were labeled (Fig. 7). These are probably incompletely digested T17 and T17b, which include amino acids 131-145 ((C)SLSADGMLTFSGPK), containing three serines as potential phosphorylation sites. There was no evidence for the labeling of T17a, which contains serine 122 and eluted earlier. Another experiment in which A was autophosphorylated in the presence of deoxycholate produced the same chromatographic profile as A labeled in the absence of deoxycholate (data not shown), indicating that autophosphorylation in the presence of deoxycholate is serine-specific as it is in its absence (41) and results in the same phosphorylated peptides produced in the absence of deoxycholate. No radioactive peptides were obtained by HPLC chromatography in the third test in which the autophosphorylated A-crystallin was not aminoethylated (data not shown). Since aminoethylation is required for solubilization of T17, this result is consistent with the autophosphorylated amino acid(s) being on T17b in this experiment.


Figure 7: Elution profile of tryptic peptides resulting from digestion of autophosphorylated A-crystallin. The peptides were fractionated by reversed-phase HPLC as described under ``Experimental Procedures.'' Closed circles indicate radioactivity of corresponding fractions; lines indicate absorbance values. Peaks 1 and 2 contain 150,000 and 400,000 cpm, respectively, and are indicated by asterisks.



Tetramerization (Deoxycholate) Has No Effect on B-Crystallin Autophosphorylation

Since deoxycholate reduces the multisubunit B-crystallin complexes to tetramers (see above), it was used to test whether aggregate size affects the autophosphorylation of purified bovine B-crystallin as it does A-crystallin (Fig. 8). The presence of deoxycholate, which activates the autophosphorylation of A-crystallin, had no positive effect on the autophosphorylation of B-crystallin (lanes 2 and 7). Neither the slight inhibition by deoxycholate (compare lanes 6 and 7) nor the weak stimulation by CHAPS (lane 8) and n-octyl glucopyranoside (lane 9) of autophosphorylation of B-crystallin were consistently reproducible. As reported previously(41) , and unlike its modest enhancement of A autophosphorylation, SDS did not stimulate the autophosphorylation of B-crystallin (lanes 5 and 10).


Figure 8: Deoxycholate does not increase B-crystallin autokinase activity. B-crystallin (10 µg) was incubated in the absence (lanes 1 and 6) or presence of 1% deoxycholate (lanes 2 and 7), CHAPS (lanes 3 and 8), n-octyl glucopyranoside (lanes 4 and 9), and SDS (lanes 5 and 10), in a reaction mixture containing 20 mM imidazole, pH 7.4, with 50 µM [-P]ATP, specific activity 1.6 Ci/mmol for 1 h at 37 °C. Autophosphorylation was monitored by SDS-PAGE and autoradiography. Lanes 1-5 are the Ponceau S-stained blot, and lanes 6-10 the corresponding autoradiograph. Densitometric values for each sample are given relative to B + deoxycholate. The positions of B-crystallin and molecular mass markers are indicated.



cAMP-dependent Kinase Phosphorylates B- More Efficiently Than A-Crystallin

To further investigate possible phosphorylation differences between A- and B-crystallin subunits, each was reacted with cAMP-dependent kinase (Fig. 9). When 10 µg of each crystallin subunit were treated with incrementally smaller amounts of bovine heart cAMP-dependent protein kinase in the presence of 20 µM cAMP, it was found that 20 ng of kinase phosphorylated B (lanes 5 and 7) at least 10 times more efficiently than A (lanes 6 and 8). Equal amounts of phosphorylation were obtained with amounts of kinase greater than 500 ng (data not shown). Several bands (lanes 7 and 8) migrating below the major A and B bands were also labeled. These radioactive bands are barely visible by staining and probably represent A and B degradation products. Shown for comparison are autophosphorylation reactions using the same amounts of B (lanes 1 and 3) and A (lanes 2 and 4) in the presence of 1% deoxycholate. It is noteworthy that A was labeled to a much higher extent by autophosphorylation in the presence of deoxycholate than by treatment with 20 ng of cAMP-dependent kinase (compare lanes 4 and 8).


Figure 9: cAMP-dependent kinase phosphorylates B- more efficiently than A-crystallin. B (10 µg) (lanes 5 and 7) or A (10 µg) (lanes 6 and 8) were incubated with 20 ng of bovine heart cAMP-dependent kinase for 30 min at 37 °C in a reaction mixture containing 20 µM cAMP, 20 mM imidazole, pH 7.4, with 50 µM [-P]ATP, specific activity 1.6 Ci/mmol. Shown for comparison are B (10 µg) (lanes 1 and 3) or A (10 µg) (lanes 2 and 4) incubated in the absence of cAMP-dependent kinase and the presence of 1% deoxycholate.




DISCUSSION

The present results show that tetramers of A-crystallin have much higher autophosphorylation activity than do endogenous high molecular weight aggregates. Deoxycholate (but not CHAPS or n-octyl glucopyranoside) disaggregates recombinant or purified bovine A from 300-1000-kDa aggregates to 80-kDa tetramers that autophosphorylate 10 times more actively than the original aggregates. The CD results showed that 1% deoxycholate had little or no effect on the secondary and tertiary structure of A-crystallin, supporting the hypothesis that tetramerization and not conformational changes are responsible for the increase in autophosphorylation activity. Consistent with this, A2D, a recombinant form of A lacking amino acids 1-63 and shown to exist as a tetramer(42) , is almost as active for autophosphorylation in the absence of deoxycholate as intact A in the presence of deoxycholate. The A2D results also indicate that increased A autophosphorylation in the presence of deoxycholate is the result of tetramerization and not chemical enhancement by deoxycholate. It is noteworthy that a theoretical model of the oligomeric structure of -crystallin based on tetrameric building blocks has been recently advanced(43) .

A relationship between disaggregation and phosphorylation has been established for several heat shock proteins which, like -crystallin, form high molecular weight aggregates. These include the small heat shock protein HSP 25/27, which disaggregates upon in vivo phosphorylation(44) , and the immunoglobulin heavy chain binding protein BIP/GRP78, which disaggregates upon in vitro autophosphorylation(45) . Phosphorylation and supermolecular organization have been shown to abolish the ability of HSP 25 to inhibit actin polymerization(46) .

Comparable to levels reported for other autophosphorylation reactions (47) , the proportion of autophosphorylated -crystallin subunits does not exceed 3%(41) . Thus, autophosphorylation of -crystallin is most likely a self-limiting reaction restricted to a small population of -crystallin polypeptides. Indeed, bovine -crystallin has been recently demonstrated to exist in at least two subpopulations that differ in shape and are not interconvertible(48) . Interaction between total bovine -crystallin and ATP has been characterized by equilibrium binding studies, tryptophan fluorescence, and P NMR(49, 50) . These studies indicated binding of ATP to -crystallin at a ratio of one ATP to two -crystallin subunits with an affinity constant of 8.1 10M. Autophosphorylation of -crystallin may also be limited by other covalent modifications. One of these is serine/threonine-specific addition of O-linked N-acetylglucosamine (O-GlcNAc) by glycosyltransferase(51) . O-GlcNAc has been mapped to serine 162 in bovine A-crystallin which is located in a region shown to be phosphorylated(36) , raising the possibility that the cAMP-dependent kinase, glycosyltransferase, and autophosphorylation reactions may compete for modification of specific serines in vivo.

The small proportion of autophosphorylated -crystallin polypeptides is one of the principal difficulties we have in unequivocally identifying the autophosphorylated A serine(s). Although we have yet to identify the exact A autophosphorylation site(s), our data clearly rule out the involvement of serine 122 and suggest the involvement of at least two phosphorylated peptides. Chicken A-crystallin, which lacks the Ser-122 cAMPdependent kinase recognition site of bovine A, exhibits autophosphorylation in the presence of deoxycholate, excluding Ser-122 as an autophosphorylation site in this species. This finding suggests that autophosphorylation may be an important mechanism for A phosphorylation that is conserved between species. Our analysis of tryptic peptides by reversed-phase HPLC ruled out the involvement of serine 122 in bovine A autophosphorylation and indicated the involvement of two peptides containing amino acids 131-145. Three serines in addition to Ser-122 between amino acids 122 and 173 have been reported to be phosphorylated in bovine A in vivo(34) . Although serines 122 and 148 are contained in the R/KXXS cAMP-dependent kinase recognition motif, they are also contained in the sequence XP(S/T)X which is a partial recognition motif for the mitogen-activated protein kinases(52) . This partial mitogen-activated protein kinase motif is shared by bovine A serines 169 and 172, the phosphorylated penultimate serine of B2(53) , and phosphorylated serines 45 and 59 of bovine B-crystallin(36) . Thus, one may speculate that in addition to cAMP-dependent kinase phosphorylation and autophosphorylation, mitogen-activated protein kinases or yet unknown kinases specific for this sequence may also be involved in crystallin phosphorylation. HSP 25/27, which is closely related to the -crystallins(24) , has been demonstrated to be phosphorylated at two S6 kinase II-like recognition sites (54) by mitogen-activated protein kinase activated protein kinase 2(55, 56) . It has also been shown that HSP 25/27 is phosphorylated in a protein kinase cascade that involves an upstream activator, an HSP 25/27 enhancer, and an HSP 25/27 kinase (57, 58) .

Our data show that A- and B-crystallin polypeptides have pronounced differences in their ability to autophosphorylate and be phosphorylated by exogenous protein kinase(s). Unlike A-crystallin, tetramerization of B in the presence of deoxycholate had no effect on autophosphorylation. Moreover, B-crystallin was phosphorylated to a higher degree than A-crystallin when each was treated with low levels of cAMP-dependent kinase. These data suggest the possibility that functional differences may be associated with the phosphorylation of A- and B-crystallin polypeptides. For example, A, but not B, has been localized to the lens membrane (59) where autophosphorylation could regulate its ability to associate with yet unidentified proteins. Another difference between A and B is that only the latter is a substrate for transglutaminase(60) . These differences may account for the fact that only B-crystallin is present in numerous non-lens tissues and is overexpressed in a variety of diseases (see (19) and (21) for additional references).

In summary, our data provide structural requirements for -crystallin autophosphorylation and demonstrate differences between A and B with respect to their phosphorylation properties. Further studies are required to explore whether these differences are related to the more specific expression of A-crystallin in the lens and the ubiquitous expression of B-crystallin in numerous normal and diseased tissues. In any event, defining the requirements for -crystallin autophosphorylation is a first step toward understanding how autophosphorylation might regulate the structure and function of this shsp/chaperone protein.


FOOTNOTES

*
This work was supported in part by Grant EYO9683 (to W. W. de J.) and by Merit Award EY03879 and a Research to Prevent Blindness Senior Investigator Award (to J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed.

The abbreviations used are: FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We wish to acknowledge the expert technical assistance of Lin Lin Ding.


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