(Received for publication, September 19, 1994; and in revised form, December 15, 1994)
From the
The 94-kDa glucose-regulated protein (endoplasmin, grp94) is an
abundant member of the 90-kDa molecular chaperone family in the
endoplasmic reticulum. We have found earlier that the 50% homologous
90-kDa heat shock protein, hsp90, has ATP-binding site(s) and
autophosphorylating activity (Csermely, P., and Kahn, C. R.(1991) J. Biol. Chem. 266, 4943-4950). In the present paper we
demonstrate that highly purified grp94 is also able to
autophosphorylate itself on serine and threonine residues. grp94 can be
freed from the co-purifying casein kinase II by concanavalin A affinity
chromatography, and its phosphorylation is unaffected by activators and
inhibitors of numerous protein kinases known to associate with the
homologous hsp90. The autophosphorylation persists in
immunoprecipitates and in SDS-polyacrylamide gel-purified and renatured
grp94. Autophosphorylation displays a monomolecular kinetics, is
activated by micromolar calcium concentrations, has an extreme heat
stability, and can utilize both ATP and GTP with relatively high k values of 243 ± 14 µM and 116 ± 23 µM, respectively. Sequence
analysis of grp94 shows the presence of two ATP-binding sites. The
major product of limited proteolysis of grp94 by chymotrypsin or papain
is an N-terminal 85-kDa fragment that can bind to ATP-agarose but does
not show autophosphorylation. Our data suggest that grp94 has an
enzymatic function analogous in many respects to the similar activity
of hsp70, hsp90, and grp78 (BiP). Autophosphorylation may participate
in/regulate the complex formation of these proteins, so it may be
involved in their chaperone function.
Exposure of cells to glucose starvation and calcium ionophores
stimulates the synthesis of a specific set of proteins localized within
the mitochondria, endoplasmic reticulum, and Golgi apparatus. These
glucose-regulated proteins can be divided into two groups showing
extensive homology with the 70- and 90-kDa heat shock protein families.
The most abundant glucose-regulated protein, grp78 ()(BiP),
and grp94 (endoplasmin, ERp99, gp96, hsp100, hsp108) are the major
representatives of these two grp
classes(1, 2, 3) .
Recent studies began to elucidate the cellular function of grp78 and grp94. Both proteins were shown to bind the immunoglobulin heavy chain in a sequential manner, and their possible involvement in chaperoning of secretory proteins was also suggested(4, 5) . grp94 (hsp100) was also shown to associate with actin filaments(6, 7) , and the human homologue of grp94, gp96, was identified as a tumor rejection antigen possibly involved in the peptide loading of the MHC I complex(8, 9) .
The exact mechanism of the interaction of glucose-regulated proteins with their targets is unknown. grp78-related chaperone effects have been shown to be ATP-dependent. ATP is translocated to the lumen of the endoplasmic reticulum (10) and binds to grp78(11, 12) . ATP converts grp78 oligomers to the monomeric form (13) and dissociates grp78 from the immunoglobulin heavy chains(14) . The maturation of immunoglobulins can be blocked either by depleting the cellular ATP levels or by mutations at the grp78 ATP-binding site(15, 16) . grp78 is able to autophosphorylate itself on Thr-229(16, 17) .
In an earlier study we
demonstrated that hsp90, a 50% homologous cytoplasmic counterpart of
grp94, possesses ATP-binding site(s) and the ability to phosphorylate
itself on serine residue(s)(18) . hsp90 was shown to undergo
large conformational changes after ATP addition(19) , and
trypanosome hsp90 seems to display an ATPase activity(20) . ATP
binding of grp94 was also demonstrated(10, 21) . These
findings raised the possibility that grp94 is also able to
autophosphorylate itself. In the present paper we verified this
hypothesis, showing a self-induced transfer of the -phosphate of
ATP or GTP to serine and threonine residues of grp94, and demonstrated
the presence of two ATP-binding sites on the protein.
Autophosphorylation of grp94 may participate in/regulate the complex
formation of this protein, (
)so autophosphorylation may be
involved in the chaperone function of grp94.
grp94 was purified both from the mouse
lymphoma cell line, L5178Y, and from livers of 4-month-old male
Sprague-Dawley rats with sequential column chromatography steps
including DEAE-cellulose DE52, hydroxyapatite, Sephacryl S-300 gel
filtration, and Mono Q fast protein liquid chromatography, as described
previously(7) . To remove the traces of co-purifying casein
kinase II, grp94 was further purified by concanavalin A-Sepharose
affinity chromatography. The ConA-Sepharose column was equilibrated
with a buffer containing 20 mM TrisHCl, 20 mM NaCl, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, pH
7.4. Bound proteins were washed with the same buffer containing 1 M NaCl. grp94 was eluted with a linear gradient (0-1,000
mM) of
-methyl-D-mannoside. The fractions
containing grp94 were collected, concentrated using a Mono Q fast
protein liquid chromatography column with a 100-1,000 mM linear gradient of NaCl in a buffer of 50 mM Tris
HCl, 2 mM EDTA, 1 mM dithiothreitol,
10% glycerol, pH 7.4, and dialyzed against a buffer containing 20
mM Tris
Cl, 100 mM NaCl, 0.1 mM dithiothreitol, 10% glycerol, pH 7.4. ConA-Sepharose-purified
grp94 proved to be essentially homogeneous as judged by SDS-PAGE
followed by silver staining.
10 µl of the immunocomplex beads was incubated in a buffer
containing 30 mM Hepes, 400 kBq of 0.2 mM [-
P]ATP, pH 7.5, in the presence of 5
mM CaCl
or MgCl
at 37 °C for 30
min with occasional mixings. The reaction was terminated by adding
Laemmli SDS sample buffer (24) and boiling for 5 min. The
proteins eluted from the immunoaffinity resins were analyzed by
SDS-PAGE (24) and autoradiography.
Figure 3:
Phosphorylation of grp94 and hsp90
immunoprecipitates (panelA), V8 peptide map of
phospho-grp94 (panelB), phosphorylation of
gel-purified grp94 and hsp90 (panelC), and
phosphoamino acid analysis (panel D). Panel A,
phosphorylation of grp94 immunoprecipitates. 2 mg of mouse Hepa-1 cell
lysate proteins was immunoprecipitated by preimmune serum (lanes3 and 4), polyclonal anti-grp94 (lanes1 and 6), or anti-hsp90 (lanes2 and 5) antibodies as described under ``Materials and
Methods.'' grp94 or hsp90 absorbed to protein G-Sepharose beads
was incubated in the presence of 5 mM CaCl (lanes1-3) or MgCl
(lanes 4-6) and 0.2 mM [
-
P]ATP in 50 mM Hepes
buffer, pH 7.4, at 37 °C for 30 min. Samples were subjected to
SDS-PAGE (H.C., immunoglobulin heavy chain). Panel B,
V8 phosphopeptide maps of autophosphorylated and casein kinase
II-phosphorylated grp94. 30 µg of ConA-Sepharose-purified grp94 was
labeled with [
P]phosphate in the presence of 5
mM CaCl
or MgCl
plus 1 µg of
purified casein kinase II as indicated. The V8 peptide map was obtained
as described under ``Materials and Methods.'' Panel
C, phosphorylation of gel-purified grp94 and hsp90.
ConA-Sepharose-purified grp94 and hsp90 were subjected to
autophosphorylation in the presence of 5 mM CaCl
or MgCl
as indicated (Control). Separate
samples were further purified by SDS-PAGE. grp94 and hsp90 bands were
cut from the gel, and after renaturation grp94 or hsp90 were
phosphorylated ``in situ'' in the gel as described
under ``Materials and Methods.'' Phosphorylated proteins were
subjected to a second SDS-PAGE to remove any associated
[
-
P]ATP (Gel-purified). Panel
D, phosphoamino acid analysis of grp94. Phosphorylation and
phosphoamino acid analysis of 6 µg of ConA-Sepharose-purified grp94
was performed in the presence of 5 mM CaCl
or
MgCl
as described under ``Materials and
Methods.'' Autoradiograms are representative of three (in case of panelB, nine) separate
experiments.
Figure 1: Removal of co-purifying casein kinase II from grp94 by concanavalin A affinity chromatography. grp94 was purified as described earlier (7) with minor modifications detailed under ``Materials and Methods.'' Afterwards the Mono Q column grp94 was further purified on concanavalin A-Sepharose as described under ``Materials and Methods.'' Fractions were analyzed for casein kinase II activity using the peptide substrate RRREEETEEE (panelA, filledcircles). Casein kinase II (CKII) content of 1.5 µg of pooled and concentrated grp94 was also measured by an intragel phosphorylation assay described earlier ((22) ; panelB).
Figure 2:
Phosphorylation of grp94 in the presence
of ATP, GTP, and various divalent cations. 1.5 µg of
ConA-Sepharose-purified grp94 was incubated in 50 mM Hepes
buffer, pH 7.4, at 37 °C for 20 min in the presence of 5 mM each of CaCl, MgCl
, and MnCl
and 0.1 mM [
-
P]ATP/GTP as
indicated. The reaction was stopped with boiling for 5 min in Laemmli
sample buffer, and samples were subjected to SDS-PAGE. The
autoradiogram is representative of three separate
experiments.
grp94 was immunoprecipitated from mouse Hepa-1 cell lysates by a
rabbit polyclonal anti-grp94 antibody. Immunoprecipitated grp94
retained its ability to incorporate [P]phosphate
from ATP in the presence of CaCl
even after extensive
washing of the immunoprecipitates, which further suggests that the
phosphorylation is an intrinsic property of grp94 and provides an easy
method to assess grp94 autophosphorylation from whole cell lysates. The
autophosphorylation of hsp90 immunocomplexes is also shown for
comparison (Fig. 3A).
hsp90, a homologous cytoplasmic
counterpart of grp94, has an extremely high tendency to associate with
other proteins, with protein kinases (18) among others.
Therefore, in spite of the apparent homogeneity of our grp94
preparation, we analyzed whether inhibitors or activators of protein
kinases known to interact with hsp90 affect the phosphorylation of
grp94. Phosphorylation of ConA-Sepharose-purified grp94 in the presence
of Ca or Mg
was not significantly
affected by 5 µg/ml heparin, 10 µM hemin, 100
µM H-7, and 100 µM H-8, which modify the
activity of casein kinase II, heme-regulated eIF-2-
kinase,
protein kinase C, and cyclic nucleotide-dependent protein kinases,
respectively. 50 µg/ml double-stranded DNA, an activator of the
double-stranded DNA-activated protein kinase did not influence the
magnesium-dependent phosphorylation of grp94; however, it induced a 90%
inhibition of the phosphate transfer in the presence of calcium-ATP
(data not shown). Lysine-rich histones (type III-S, Sigma) or histone
H1 induced an 8-10-fold increase in the magnesium-dependent
phosphorylation of grp94. On the contrary, they did not influence the
calcium-dependent phosphorylation of the protein similarly to the
effects of lysine-rich histones on the phosphorylation of hsp90 ( (18) and data not shown).
To gain further evidence that the
calcium-dependent phosphorylation is not caused by the presence of
enzymatically and immunologically undetectable traces of casein kinase
II, we compared the V8 peptide maps of P-labeled
ConA-purified grp94 phosphorylated in the presence of CaCl
or MgCl
without and with exogenous casein kinase II,
respectively. The phosphorylation pattern of the two peptide maps was
clearly different, providing further evidence that the
calcium-dependent phosphorylation of grp94 was not caused by traces of
contaminating casein kinase II (Fig. 3B).
To analyze further whether the phosphorylation of grp94 is an intrinsic property of the protein we purified ConA-Sepharose-purified grp94 using SDS-polyacrylamide gel chromatography. After SDS-PAGE and renaturation the calcium-dependent phosphorylation of grp94 still persisted. However, the magnesium-dependent phosphorylation of grp94 was significantly diminished after gel purification of the protein, similarly to that of hsp90 (Fig. 3C).
Phosphoamino
acid analysis of phospho-grp94 revealed that the phosphorylation of the
protein resulted in the transfer of
[-
P]phosphate of ATP to serine and
threonine residues in the presence of MgCl
or CaCl
(Fig. 3D).
Figure 4:
Kinetics (panelA), heat
stability (panelB), ion (panelC),
and pH (panelD) dependence of grp94
autophosphorylation in comparison with the pH dependence of hsp90
autophosphorylation (panelE). PanelA, kinetics of grp94 autophosphorylation. 10 µg of
ConA-purified grp94 was phosphorylated in the presence of 5 mM CaCl after setting the final concentration of the
protein as indicated under ``Materials and Methods.'' Samples
were concentrated in a SpeediVac centrifuge evaporator prior to
SDS-PAGE. PanelB, heat stability of grp94
autophosphorylation. 1.5 µg of ConA-Sepharose-purified grp94 was
incubated at the temperatures indicated for 10 min in 50 mM Hepes, pH 7.4. Samples were cooled to 37 °C and phosphorylated
as described under ``Materials and Methods.'' The control
(100%) values of magnesium-dependent autophosphorylation (filled
circles) and calcium-dependent autophosphorylation (open
circles) were 0.4 and 0.2 nmol of
P/min
mg of
grp94, respectively. PanelC, ion dependence of grp94
autophosphorylation. Autophosphorylation of grp94 was performed as
described under ``Materials and Methods'' in the presence of
CaCl
or MgCl
at final free concentrations
indicated. Free concentrations of micromolar Ca
were
set using a calcium-EGTA buffer(53) . Open and filledcircles represent magnesium- and
calcium-dependent autophosphorylation, respectively. Data are means
± S.D. of the densitometric analysis of autoradiograms from
three separate experiments. PanelsD and E,
pH dependence of the autophosphorylation of hsp90 and grp94,
respectively. Rat liver hsp90 and ConA-Sepharose-purified grp94 were
autophosphorylated as described under ``Materials and
Methods'' in the presence of 5 mM CaCl
or
MgCl
. The pH of the reaction medium was set using 50 mM MES (pH range 5-6.5), Hepes (pH range 6.5-8), and Tris
(pH range 8-10). Autophosphorylation of hsp90 and grp94 was not
significantly different in different buffers at overlapping pH values.
Data represent means ± S.D. of the densitometric analysis of
autoradiograms from three separate
experiments.
Autophosphorylation of grp94 displays an extreme heat stability in the presence of calcium. The amount of grp94-incorporated radioactive phosphate is essentially unchanged even after an incubation of 10 min at 95 °C (Fig. 4B). However, after boiling for 5 min, the autophosphorylation of grp94 is greatly diminished to 30-40% of the control value (data not shown). The magnesium-dependent phosphorylation is much more sensitive for heat denaturation, losing half of its activity at about 55 °C. In spite of this higher heat sensitivity, approximately 25% of the original magnesium-dependent activity remains stable even after a heat treatment at higher temperatures (Fig. 4B).
Autophosphorylation of grp94 occurs at micromolar free calcium
concentrations, reaching a plateau after 10 µM Ca (Fig. 4C). The
magnesium-dependent phosphorylation has a sharply different pattern,
activated by only millimolar concentrations of Mg
and
declining after 20 mM divalent cation (Fig. 4C). Addition of 100 mM NaCl or KCl to
the reaction medium induces a slight (approximately 20%) inhibition of
both the calcium- and magnesium-dependent phosphate transfer (data not
shown).
The pH dependence of the calcium- and magnesium-dependent phosphorylation of grp94 shows a broad optimum peaking around pH 7.5 and 9.0, respectively (Fig. 4D). On the contrary the pH dependence of calcium- and magnesium-dependent phosphorylation of hsp90 is dissimilar, rendering the calcium- and magnesium-dependent phosphorylation predominant at about pH 6.0 and 7.0, respectively (Fig. 4E).
The k values of the
autophosphorylation of grp94 for ATP in the presence of CaCl
or MgCl
were 243 ± 14 µM and 111
± 14 µM, respectively. The respective k
values for calcium-GTP and magnesium-GTP were
significantly lower, 116 ± 23 µM and 20 ± 4
µM (data not shown).
Figure 5: Autophosphorylation of grp94 after (panel A) and before (panel B) limited proteolysis with chymotrypsin and papain. PanelA, autophosphorylation of grp94 after limited proteolysis; panelB, autophosphorylation of grp94 before limited proteolysis. Limited proteolysis and calcium-dependent autophosphorylation of 1.5 µg of ConA-Sepharose-purified grp94 were performed as described under ``Materials and Methods.'' Samples were subjected to SDS-PAGE, and the radioactivity of the proteolytic fragments was analyzed by autoradiography. Autoradiograms are representative of two separate experiments.
When applied to an ATP-agarose column, both grp94 and its 85-kDa proteolytic fragment were retained, suggesting the presence of a functional ATP-binding site in the 85-kDa fragment (in control experiments with agarose microcolumns both proteins were in the flow-through fraction (data not shown). Microsequencing the 85-kDa fragment gave an N terminus of DDEVD, which completely matches the processed N-terminal sequence of mouse grp94(30) . (There was an agreement between the sequence of the 85-kDa fragment and that of grp94 at the consecutive 8 amino acid residues as well; data not shown.) These results reflect that both papain and chymotrypsin truncate grp94 at its C terminus.
In an earlier study we demonstrated that hsp90, an approximately 50% homologous cytoplasmic counterpart of grp94, possesses ATP-binding site(s) and is able to phosphorylate itself on serine residue(s) (18) . grp94 was also shown to bind ATP(10, 32) . These findings raised the possibility that grp94 is also able to autophosphorylate itself.
Our experiments
revealed that highly purified preparations of grp94 can incorporate
radiolabeled phosphate from the position of both ATP and GTP.
Control experiments with
-
P-labeled ATP as well as
the detection of the radiolabel on serine and threonine residues after
phosphoamino acid analysis strongly suggest that the
-phosphate
was transferred to grp94.
We detected a significant amount of casein kinase II co-purifying with apparently homogenous grp94 preparations. This is not surprising, since the homologous hsp90 tightly associates with a number of protein kinases including casein kinase II(18, 22) , and grp94 is a good substrate of casein kinase II(33) . The complexing of grp94 with casein kinase II may occur in vivo, since grp94 was reported to be associated with the nucleus(34) , and casein kinase II, a predominantly nuclear protein kinase, was also reported to be present in the endoplasmic reticulum(32) . Further studies are necessary to elucidate whether this is indeed the case or whether grp94 sticks to casein kinase II during the isolation procedure.
Because of the tight association of grp94 with casein kinase II extreme care should be exercised to preclude the possibility that the phosphorylation of purified grp94 was induced by traces of contaminating casein kinase II. In our studies we obtained several lines of evidence against this possibility. 1) Casein kinase II can be efficiently removed from grp94 by ConA affinity chromatography and high salt wash of the latter protein. ConA-purified grp94 contains no casein kinase II detected by enzymatic analysis using two appropriate substrates, the peptide RRREEETEEE, and dephosphorylated casein. 2) ConA-purified grp94 does not contain any immunodetectable casein kinase II, and immunodepletion of putative traces of casein kinase II does not diminish the calcium-dependent phosphorylation of grp94. 3) The phosphorylation pattern of autophosphorylated grp94 clearly differs from that of the casein kinase II-phosphorylated protein. 4) Calcium-dependent phosphorylation of grp94 cannot be inhibited by heparin, a sensitive inhibitor of casein kinase II, and displays an extreme heat stability, which is not characteristic of casein kinase II even in its complex with hsp90, a highly homologous heat shock protein(22) .
Although casein kinase II activity can be efficiently and fully
removed from grp94 preparations by concanavalin A affinity
chromatography the possibility still persists that traces of other
contaminating kinases are still present in the grp94 preparation and
induce the calcium-dependent phosphorylation of the 94-kDa band.
Several lines of evidence show, however, that this is not the case. 1)
The phosphorylation of grp94 persists after immunoprecipitation of
grp94 by anti-grp94 antibodies and after further purification of the
protein on SDS-PAGE. 2) The calcium-dependent phosphorylation displays
a monomolecular kinetics. 3) The approximate k of
the reaction is 0.24 mM for ATP. This is much higher than the
reported values for most other protein kinases. Furthermore,
phosphorylation is not affected by a number of activators and
inhibitors of the protein kinases that might associate with grp94. 4)
The phosphorylation displays a unique cation dependence, being active
in the presence of Ca
ions alone. 5) The activity is
surprisingly heat-stable. grp94 retains its autophosphorylation
activity almost fully even after incubation for 10 min at 95 °C.
Thus, these data strongly suggest that grp94 itself possesses an intrinsic, calcium-dependent autophosphorylating activity. Many of the arguments listed above are also valid for the magnesium-dependent phosphorylation of grp94. However, the magnesium-dependent phosphorylation displays a much smaller resistance against heat treatment than its calcium-dependent counterpart, and the extent of magnesium-dependent phosphorylation is significantly diminished after gel purification of grp94. On one hand these differences may reflect the involvement of remote amino acid side chains in the active center of grp94 phosphorylation in the presence of magnesium, which are parts of protein segments being more sensitive for heat denaturation and do not completely refold after guanidinium chloride treatment. On the other hand we cannot exclude the possibility that the magnesium-dependent phosphorylation of grp94 occurs as a result of a trace amount of contaminating protein kinase, which is not similar to those kinases known to be associated with hsp90.
Dechert et al.(35) have isolated an 80-kDa protein kinase from the microvessels of porcine brain. The protein had an N-terminal amino acid sequence similar to that of hsp108, a chicken homologue of grp94(35, 36) . Our findings further suggest that the 80-kDa protein of Dechert et al.(35) is indeed a grp94 homologue displaying a similar enzymatic activity. During the preparation of our manuscript Li and Srivastava (9) reported that the structure of grp94 contains an ATP-binding site and that the protein displays an ATPase activity similar to that of hsp90. In our studies we also detected a significant ATPase activity of grp94, albeit significantly smaller than that reported by Li and Srivastava(9) . The ATPase activity was greatly diminished when we further purified grp94 with ConA-Sepharose affinity chromatography (data not shown). These findings substantiate the conclusion that the acceleration of phosphate transfer in highly purified grp94 preparations is an intrinsic property of grp94.
Comparing the primary structure of mouse(30) , chicken(36) , and human (37) grp94 homologues with ATP binding consensus sequences(38, 39) , we found a second ATP-binding site of grp94 located toward the N-terminus from the ATP-binding consensus sequence identified by Li and Srivastava (Table 1). Interestingly the crucial GKT motif in the ATP-binding site of hsp90 (18) was conservatively mutated to GKR in grp94, which makes it very unlikely that these otherwise highly homologous proteins use the same ATP-binding sites.
Partial proteolytic digestion of grp94 with papain or chymotrypsin produced a major proteolytic fragment of 85 kDa. The identity of the N terminus of this fragment with that of grp94 suggests that under these conditions papain and chymotrypsin remove the C-terminal end of grp94, leaving its N terminus intact. Our difficulties in obtaining the N-terminal sequence of the 85-kDa fragment and our unsuccessful attempts to digest it with leucineaminopeptidase suggest that the N terminus of grp94 is blocked. These conclusions are in agreement with the earlier data of Kulomaa et al.(36) and Edwards et al.(40) . When the last 80-90 amino acid residues were removed from grp94, the autophosphorylation sites remained intact, since when autophosphorylating grp94 prior to the partial proteolytic digestion the radiolabel was recovered in the major, 85-kDa proteolytic fragment. On the other hand, the C-terminal end of the protein may be necessary for the autophosphorylation to occur. This part of grp94 contains an adenine-binding consensus sequence (amino acids 708-719 of ERp99, Table 1), which may participate in the autophosphorylation.
Autophosphorylation of grp94 requires the presence of either calcium
or magnesium ions. grp94 is reported to be a calcium-binding protein (32) possessing 4 high affinity and 11 low affinity
calcium-binding sites with apparent dissociation constants of 2 and 600
µM, respectively(34) . Examining the primary
structure of grp94 we found three conserved putative high affinity
calcium-binding sites displaying a homology with the consensus sequence
of the calcium binding EF-hand motif(41) . Among these three
sequences (amino acids 224-235, 308-319, and 440-451
in the mouse grp94 sequence (30) the second and its
surroundings had an unambiguous -helical structure, the third
EF-hand region was partially
-helical, and the first EF-hand
region contained hardly any
helical segments as predicted by the
methods of Chou and Fasman (42) and Garnier et
al.(43) . grp94 also has a number of amino acid sequences
close to its C terminus, which display a partial homology with EF-hand
structures functioning presumably as low affinity calcium-binding
sites. The presence of high affinity calcium-binding sites on grp94
provides a structural and functional explanation of the activation of
its autophosphorylation by micromolar concentrations of calcium.
Comparing the characteristics of grp94 autophosphorylation with
hsp90, grp78 (BiP), and hsp70 (DnaK) autophosphorylation, we found that
both glucose-regulated proteins, grp94 and grp78, residing in the lumen
of the endoplasmic reticulum show a high, micromolar affinity for
calcium, whereas their cytoplasmic counterparts, hsp90 and hsp70, are
activated only in the presence of millimolar concentrations of the
cation (Table 2). On the contrary, cytoplasmic and luminal free
Caconcentrations are generally assumed to be
approximately 0.1 and 100 µM, respectively(44) .
Intracellular calcium concentration is reported to be increased after
various environmental stresses(45) , and depletion of the
calcium stores of the endoplasmic reticulum may contribute to the
activation/synthesis of grp78(46, 47) . Further
studies are required to elucidate whether stress-induced local
perturbations in the cytoplasmic and luminal calcium concentration may
induce the activation of hsp90 and grp94 autophosphorylation,
respectively.
Although to the best of our knowledge the luminal free
Mg concentration has never been exactly measured, it
is assumed to be in the millimolar range(48) . The sharp
increase in the magnesium-dependent autophosphorylation of grp94 makes
the luminal Mg
concentration a good candidate for the
regulation of grp94, similar to grp78(47) .
Heat shock and
other stresses cause a drop in the intracellular pH and ATP
concentration(49) . Whereas stress-induced intracellular
acidification may activate the autophosphorylation of hsp90, it is
hardly playing any role in the regulation of grp94. On the contrary,
the relatively high k for ATP makes both hsp90 and
grp94 sensitive to respond to greater changes in intracellular ATP
concentration after various stresses, e.g. ischemia.
The
stoichiometry of the autophosphorylation is rather low, reaching
2-6% under regular (suboptimal) assay conditions. Incubating the
protein at a higher temperature (50-60 °C) for longer times
(30-60 min) increased the extent of autophosphorylation. However,
the calcium-dependent autoproteolysis of grp94 ()competed
with the reaction and prevented the full analysis of the stoichiometry
of the reaction (data not shown). A low level of autophosphorylation
(2-15%) is a characteristic feature of all heat shock proteins (Table 2). Autophosphorylation may accompany the
association/dissociation of heat shock proteins with their targets,
which may be rate-limiting.
The autophosphorylation of
grp94 may occur only transiently in vivo. grp94 is reported to
be a phosphoprotein only in some cell types(32, 34) .
This may indicate that its autophosphorylation sites are not constantly
occupied in vivo. The presence of phosphoprotein phosphatases
and the transfer of the -phosphate from serine and threonine
residues of grp94 to water via an ATPase reaction may explain why
Clairmont et al.(10) did not get a significant
thiophosphorylation of grp94 in intact canine pancreas microsomes.
Recent studies indicate that the autophosphorylation of grp78 may also
occur only transiently in vivo since its autophosphorylation
site, Thr-229, is usually found nonphosphorylated in isolated
grp78(17) .
Since the magnesium-dependent
autophosphorylation of grp94 is greatly enhanced by lysine-rich
histones, e.g. by histone H1, and grp94 was reported to be
enriched in the cell nucleus after heat shock (34) , the
autophosphorylation of grp94 may be involved in the protection of
nuclear structures after environmental damage. The autophosphorylation
of grp94 may also play a role in the dissociation of grp94 from other
proteins in the analogy of similar effects on hsp60 (50) ,
grp78(12, 13, 14) , and
hsp90(51) . This may reflect an involvement of
grp94 autophosphorylation in the chaperoning of secretory proteins and
in the peptide loading of the MHC I complex (8, 9) .
Addendum-While our paper was under review, Dechert et al.(54) published the full sequence of the grp94-related protein kinase of porcine brain showing a 92-98% homology with grp94. Their results are consistent with our findings on the autophosphorylation of grp94 and raise the possibility that the protein is able to phosphorylate substrates other than itself.