(Received for publication, December 23, 1994)
From the
Previously we showed that
The two
The 173-amino-acid
Extracted
Figure 1:
Deoxycholate converts
Figure 2:
A, far UV CD spectra of
Figure 3:
Deoxycholate increases autokinase activity
of recombinant
Figure 4:
Specificity of autophosphorylation in the
presence of deoxycholate. Recombinant
Autophosphorylation of
Figure 5:
The effect of deoxycholate on
autophosphorylation by
Figure 6:
Chicken
Figure 7:
Elution profile of tryptic peptides
resulting from digestion of autophosphorylated
Figure 8:
Deoxycholate does not increase
Figure 9:
cAMP-dependent kinase phosphorylates
The present results show that tetramers of
A relationship between disaggregation and
phosphorylation has been established for several heat shock proteins
which, like
Comparable to levels reported for other
autophosphorylation reactions (47) , the proportion of
autophosphorylated
The small proportion of autophosphorylated
Our data show that
In summary, our data provide structural requirements
for
We wish to acknowledge the expert technical assistance
of Lin Lin Ding.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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) .
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) .
-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.
Preparation of
Bovine -Crystallin
Polypeptides
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
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.
A-Crystallin
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
[
1-2 mg of recombinant P]
A-Crystallin Tryptic
Peptides
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) .
Deoxycholate Disaggregates the
The
aggregation state of A- and
B-Crystallin Multisubunit Complexes to 80-kDa Tetramers
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).
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
Fig. 2A shows the
far UV circular dichroism (CD) spectra of recombinant A-Crystallin
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.
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
The relative amounts of autophosphorylation of
recombinant A-Crystallin Is Enhanced by
Tetramerization
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).
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).
A (10 µg, re
A) (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
Previous work demonstrated that
deletion of amino acids 1-63 of bovine A-Crystallin
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.
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).
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
The major in vivo phosphorylation
site for bovine A-Crystallin
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.
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
Autophosphorylation of chicken A-Crystallin
by Reversed-phase HPLC Separation of Tryptic
Peptides
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.
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
Since deoxycholate reduces
the multisubunit B-Crystallin Autophosphorylation
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).
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
To further investigate
possible phosphorylation differences between B- More
Efficiently Than
A-Crystallin
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).
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.
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) .
-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) .
-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
10
M
. 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.
-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) .
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).
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.