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Advances in Brief |
Section of Molecular Therapeutics, Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [K. S. S., S. R. S. M., J. S., F. A.-O.], and the Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555 [T. K. H.]
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ABSTRACT |
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-32P]ATP resulted in
Mg2+-dependent phosphorylation of the endogenous AGT.
Immunoprecipitation after exposure of the cells to
32P-labeled inorganic phosphate showed that AGT exists as a
phosphoprotein under physiological conditions. Western analysis and
chemical stability studies showed the AGT protein to be phosphorylated
at tyrosine, threonine, and serine residues. Purified protein kinase A
(PKA), casein kinase II (CK II), and protein kinase C (PKC)
phosphorylated the recombinant AGT protein with a stoichiometry of
0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein),
respectively. Residual phosphorylation of the endogenous AGT by the PKs
present in cell homogenates and phosphorylation of the recombinant AGT
by purified serine/threonine kinases, PKA, PKC, and CK II reduced AGT
activity by 30–65%. Conversely, dephosphorylation of cell extracts by
alkaline phosphatases stimulated AGT activity. We also identified
consensus phosphorylation motifs for many cellular kinases, including
PKA and CK II in the AGT protein. These data provide the first and
conclusive evidence of AGT phosphorylation and suggest that reversible
phosphorylation may control the activity of this therapeutically
important DNA repair protein in human normal and cancer cells. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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AT transitions. With regard to
bifunctional alkylators like the chloroethylnitrosoureas, AGT prevents
the production of cytotoxic DNA interstrand cross-links by repairing
the O6-chloroethylguanine cross-link
precursors induced by these agents (1
, 5)
. Among the
pseudosubstrates developed for AGT is BG, which effectively depletes
cellular AGT and enables significant potentiation of drug-induced
cytotoxicity (7)
. BG is currently undergoing clinical
trials to improve the efficacy of chloroethylnitrosoureas against brain
tumors and other human cancers (8)
. In contrast to its
overexpression in neoplasms, AGT is expressed at very low levels in
normal tissues such as brain and bone marrow (4
, 9)
, which
increases their susceptibility to the toxic and mutagenic effects
exerted by the environmental carcinogens and anticancer drugs. Currently, the expression and biochemical regulation of AGT in human cells are poorly understood. DNA hypermethylation has been shown to repress the transcriptional activity of the AGT gene in some human tumor cell lines (10) . However, in a significant majority of human cancers, AGT is expressed at various levels, and the biochemical mechanisms that affect the AGT protein or its activity are largely unknown. The dealkylation reaction of AGT, which comprises its binding to DNA, recognition of the O6-alkylated guanine, and self-transfer of the alkyl group is a direct process that does not involve other proteins or cofactors. However, the existence of intracellular pathways that can alter AGT activity, its stability, and/or its subcellular distribution will have a significant effect on AGT function and the extent of DNA repair. Clarification of such mechanisms in cells of both normal tissues and tumors is important not only for understanding the physiological regulation of AGT but also for developing new strategies to enhance the efficacy of AGT-targeted chemotherapy. We have shown previously that the AGT protein after being inactivated by BG or 1,3-bis(2-chloroethyl)-1-nitrosourea, is targeted for polyubiquitination and subsequent degradation by the proteasome complex (11) . Because ubiquitin conjugation of many target proteins is often interlinked with phosphorylation (12 , 13) and because phosphorylation affects the DNA binding and function of many enzymes in nucleic acid metabolism including DNA repair (14, 15, 16) , we investigated protein phosphorylation as a potential regulatory mechanism for human AGT.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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-32P]ATP (3000 Ci/mmol)
were purchased from ICN Radiochemicals (Costa Mesa, CA). Bacterial and
calf intestine alkaline phosphatases were procured from Sigma.
AGT Activity Assay.
Exponentially growing UW228 cells were trypsinized and washed with
Tris-buffered saline (TBS; pH 8.0). Cell extracts were prepared by
sonication in TGED buffer [40 mM Tris-HCl (pH 8.0), 10%
glycerol, 1 mM EDTA, and 0.5 mM DTT], followed
by centrifugation at 10,000 x g for 10 min.
AGT activity was determined by quantitating the transfer of the
3H-labeled methyl group from the
O6 position of guanine in the DNA to
the AGT protein (20)
. Briefly, cell extracts (50–200 µg
of protein) were supplemented with 2 µg of DNA substrate enriched
with O6-methylguanine (
10,000 cpm)
and incubated at 37°C for 30 min, after which the DNA was hydrolyzed
in trichloroacetic acid at 80°C for 30 min. The protein precipitates
were collected on glass fiber filters and solubilized, and the
radioactivity was counted (20)
.
Combined Immunoprecipitation/Immunoblotting Analysis of AGT
Protein.
This procedure has been described elsewhere (11)
. Briefly,
cell extracts containing AGT protein (>150 µg/assay) were precleaned
with protein A-agarose beads and incubated with 2 µg of polyclonal
antibodies to human AGT. After 6 h at 4°C, immune complexes were
collected by adding protein A-agarose beads and washed three times with
TBS containing 0.1% Tween 20. The pellets were solubilized in 1% SDS
and 20 mM Tris-HCl (pH 6.8). After boiling, the samples
were electrophoresed on 12% SDS-polyacrylamide gels under nonreducing
conditions (11)
. After electrophoretic transfer of the
proteins on to Immobilon-P membranes (Millipore Co.) and blocking in
4% BSA solution, the blots were probed with the appropriate
antibodies. Positive bands were visualized by enhanced
chemiluminescence (ECL, Amersham Co.).
Phosphorylation of Endogenous AGT in Cell Extracts and Its
Influence on AGT Activity.
To study 32P incorporation, cell extracts (150
µg of protein in a volume of 100 µl) prepared in TGED buffer were
supplemented with [-32P]ATP (10
µM; 0.1 µCi) in the presence or absence of
MgCl2 (0–10 mM). After incubation at
30°C for 20 min, the reactions were terminated by adding 300 µl of
ice-cold TBS containing 10 mM EDTA, and the AGT protein
present in the extracts was immunoprecipitated as described above.
After electrophoresis of solubilized immunoprecipitates on 12%
polyacrylamide gels, the gel was dried and exposed to X-ray film to
visualize the 32P-labeled AGT protein. A
duplicate set of kinase assays with 50 µg of protein, 10
µM unlabeled ATP instead of
[-32P]ATP were processed for Western
blotting of the AGT protein.
Quantitation of the effect of phosphorylation on AGT activity was performed in two steps, i.e., in vitro phosphorylation followed by analysis of AGT activity in the phosphorylated extracts. Cell extracts in TGED buffer (50–200 µg of protein) were treated with 0.5 mM ATP and 2.5–10 mM MgCl2. Extracts with no supplements, Mg2+ alone, or with ATP alone served as controls. After incubation at 30°C for 1 h, EDTA was added at 10 mM to stop the kinase reactions. To avoid any inhibitory effects that Mg2+, ATP, EDTA, and their complexes may have on AGT activity, the assay sample volumes were adjusted to 75 µl in TGED buffer and desalted by microgel filtration by using 1-ml spin columns packed with Biogel p6 beads (Bio-Rad), according to the manufacturer’s instructions. After applying the samples, the columns were centrifuged at 3500 x g into tubes containing 50 µl of TGED buffer. Protein recovery from the spin columns was >90%. All sample volumes were adjusted to 100 µl, [3H] DNA substrate was added, and AGT activity was quantified as described above.
Dephosphorylation and AGT Activity in Cell Extracts.
UW228 cell-free extracts (50–150 µg protein) were treated with 20
units of alkaline phosphatase from Escherichia coli or calf
intestine in 30 mM Tris-HCl (pH 8.0) at room
temperature for 1 h. The phosphatases were highly active under
these conditions. Controls included cell extracts, and all reaction
components except the alkaline phosphatase. EDTA (to 1
mM) and EGTA (to 0.5 mM)
were added to inactivate the phosphatases. The samples were then
filtered on Biogel p6 columns, DNA substrate was added, and AGT
activity was quantified as described above.
Intracellular Phosphorylation of AGT.
UW228 cells cultured in 25-cm2 flasks were washed
with phosphate-deficient DMEM (Life Technologies, Inc.) and incubated
for 6 h with the same medium containing
[32P]Pi (10 µCi/ml) for 6 h. The
radiolabeling was terminated by washing the cells twice with ice-cold
TBS. After trypsinization, cells were lysed in 0.5 ml of TBS containing
0.5 mM phenylmethylsulfonyl fluoride, 50 mM
sodium fluoride, 0.5 mM sodium vanadate, and 1% NP40. The
supernatants recovered by centrifugation were immunoprecipitated with
AGT antibodies as described earlier. The immunocomplexes were resolved
on 12% SDS-polyacrylamide gels, and the gels were dried and subjected
to autoradiography.
Phosphorylation of Recombinant AGT by PKA, PKC, and CK II:
Reaction Stoichiometry and Effect on AGT Activity.
Full-length human AGT protein was expressed in E. coli BL21
(DE3, pLys) by using a T7 promoter-based expression vector, and the
protein was purified to homogeneity as described previously
(21)
. Phosphorylation of this substrate by the purified
kinases was performed according to published protocols
(22)
. To determine the stoichiometry of phosphorylation,
we suspended AGT protein (1 µg/assay) in phosphorylation buffer
containing 20 mM Tris-HCl (pH 7.4) and 2.5–10
mM MgCl2. For reaction with
PKA, 0.05% Triton X-100, 1 mM EDTA, and 2 units
of PKA were added. For reaction with PKC, 1 mM
CaCl2, 0.5 mg/ml phosphatidylserine, 10 µg/ml
diolein, and 0.5 units of PKC were added. For reaction with CK II, 25
mM KCl and 4 units of CK II were added. The
kinase amounts were chosen to maximize AGT phosphorylation. The
reactions were started by adding [-32P]ATP
(3 µCi, 0.1 mM) and terminated after 1 h
incubation at 30°C by adding the SDS-PAGE sample buffer. The samples
were electrophoresed on 15% polyacrylamide gels, the gels were stained
with Coomassie blue, and the AGT protein bands were cut and added to 5
ml of scintillation fluid for counting. A duplicate gel was dried and
autoradiographed to visualize the phosphorylated AGT.
To study the effect of the phosphorylation by PKA, PKC, and CK II on AGT activity, kinase reactions were performed under conditions similar to those described above except for the use of 0.25 mM unlabeled ATP and 4 µg of recombinant AGT protein as the substrate, and incubation time was decreased to 30 min. Controls for these reactions included all components except the PKs. The reactions were terminated by adding 5 mM EDTA, the samples were filtered on Biogel P6 spin columns, and AGT activity was assayed in the eluates as described above.
Identification of Phosphorylated Residues in the AGT Protein.
Immunoprecipitation/immunoblotting analysis (11)
and
chemical cleavage (23)
were applied to identify which
amino acids in the AGT protein are phosphorylated. In the first method,
the AGT protein was immunoprecipitated from UW228 extracts (300 µg
protein) in quadruplicate as described earlier. Solubilized
immunoprecipitates were electrophoresed and Western blotted. The
membrane strips were probed separately with monoclonal antibodies
specific to AGT (2 µg/ml), phosphotyrosine (1 µg/ml), phosphoserine
(3 µg/ml), and phosphothreonine (0.5 µg/ml).
We also examined the differential stability of phosphate groups linked to aliphatic and aromatic amino acids under acidic or alkaline conditions (23) . This procedure involved immunoprecipitating the AGT protein from 32P-labeled UW228 cells, solubilizing the immunocomplexes, and dividing into three equal portions, one of which was untreated (control), another treated with 0.2 M HCl at 60°C, and the third treated with 1 M NaOH at 75°C for 30 min. All samples were neutralized to pH 6.5 and electrophoresed on 12% SDS gels, which were then dried and subjected to autoradiography.
AGT Migration after Phosphorylation and Dephosphorylation.
Under the reaction conditions described in the previous sections,
recombinant AGT protein (1 µg) was treated with CK II, and UW228 cell
extract (60 µg) which contains constitutively phosphorylated AGT was
dephosphorylated with alkaline phosphatase. These samples were
subjected to SDS-PAGE and Western blotting to detect differences in the
mobility of the AGT protein.
Statistical Analysis.
All experiments including the effect of phosphorylation and
dephosphorylation on AGT activity were performed at least five separate
times unless otherwise indicated. Results were assessed by Student’s
t test. Significance was defined as P < 0.05.
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Results and Discussion |
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). Computer-assisted motif analyses (24
, 25) revealed the
presence of multiple, often overlapping, phosphorylation sites for the
serine/threonine PKs, PKA, CK I, CK II, and glycogen synthase kinase 3.
These sites are clustered at the extreme NH2
terminus of the AGT protein, surrounding amino acids 10–39. (Fig. 1
).
A single tyrosine phosphorylation motif encompassing residues 107–114
and an additional CK I site at the COOH terminus of the protein were
also found. Results showing that many of these PKs indeed phosphorylate
the AGT protein are described in this report.
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Phosphorylation of AGT Protein in Cell-free Extracts.
As a first step in characterizing AGT phosphorylation, we examined
label incorporation from [-32P]ATP in UW228
cell-free extracts to the endogenous AGT protein. In the absence of
Mg2+ ions, no 32P label was
transferred to the AGT polypeptide; however, this incorporation was
greatly enhanced as Mg2+ concentration increased
in the reaction (Fig. 2A
). A Western blot performed in parallel and probed with AGT
antibodies showed that equal amounts of AGT protein were present in all
samples (Fig. 2B
). These data indicate that the AGT protein
undergoes phosphorylation in vitro, and that the PK or
kinases that mediate this reaction are present intracellularly. The low
level of 32P incorporation observed in this
experiment may reflect substrate limitation attributable to
constitutive AGT phosphorylation in UW228 cells.
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). On the
basis of our previous studies (11)
, we believe that the
higher molecular weight 32P-labeled bands
observed in Fig. 2C
represent the polyubiquitinated forms of
AGT. Therefore, it is tempting to speculate that AGT phosphorylation
may precede its conjugation with ubiquitin; such a relationship between
these two posttranslational events has been demonstrated for other
proteins (12
, 13)
. The addition of purified AGT protein to
the cell extract before immunoprecipitation quenched the signal and
confirmed the identity of the radioactive immunoprecipitated protein as
AGT. Furthermore, 50% of the radioactivity associated with the AGT
was released by treatment with alkaline phosphatase. These data provide
clear evidence that AGT exists as a phosphoprotein in human glioma
cells and suggest that phosphorylation is an important biochemical
modification for this protein.
Identification of Amino Acids Phosphorylated in the AGT Protein.
We used an immunological and chemical approach to identify the
phosphogroup acceptors in the DNA repair protein. In the first
procedure, the AGT protein was purified from UW228 cell extracts by
immunoprecipitation and subjected to Western blotting in quadruplicate.
The blots were probed independently with antibodies specific to AGT,
phosphotyrosine, phosphoserine, and phosphothreonine. The specificity
of antiphosphoamino acid antibodies was verified by competition with 1
mM free phosphoamino acids. All three phosphoamino acid
antibodies reacted strongly with the AGT protein bands, indicating the
presence of serine, threonine, and tyrosine phosphorylations in the
cellular AGT protein (Fig. 3A
). These results were further confirmed by exploiting the
differential lability of the phosphate groups bound to aliphatic and
aromatic amino acids; whereas the phosphate linked to serine and
threonine is acid stable but alkali labile, O-tyrosyl
phosphate groups remain stable in both acid and alkali
(23)
. The autoradiographic patterns after the acid or
alkali treatment of AGT immunoprecipitates prepared from
32P-labeled UW228 cells are shown in Fig. 3B
. The intensity of the
32P-labeled AGT bands after acid treatment
(Fig. 3B
, Lane 1) was similar to that of the untreated
control (Fig. 3B
, Lane 2). However, alkali treatment
generated faint radioactive bands of AGT (Fig. 3B
, Lane 3),
reflecting the loss of phosphates associated with serine and threonine
and retention of the tyrosine-linked phosphate groups. Collectively,
these results demonstrate that AGT is phosphorylated at multiple sites,
and that serine, threonine, and tyrosine are the modified residues.
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), we performed similar experiments by incubating cell
extracts in the presence or absence of unlabeled ATP and
Mg2+ and then desalting them and quantitating AGT
activity. When extracts were incubated with either ATP or
Mg2+ alone, no significant changes in AGT
activity were observed. However, when ATP-Mg2+
were both present, AGT activity was consistently inhibited (Fig. 4A
). The extent of inhibition ranged from 40 to 65% in
different experiments, regardless of the protein concentration used.
Significantly, Mg2+ at the physiologically
relevant concentration of 1 mM could promote AGT
phosphorylation and therefore its inhibition.
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60% more than that of controls (Fig. 4B
).
Lower protein concentrations (in the cell extracts) and longer
incubations with the phosphatases were necessary to observe the
increases in AGT activity. The calf intestinal enzyme was more
efficient in the dephosphorylation reaction. Treatment of the cell
extracts with agarose-immobilized alkaline phosphatase also resulted in
increased AGT activity. Taken together, the findings of decreased AGT
activity after in vitro phosphorylation (Fig. 4A
)
and enhanced activity after dephosphorylation (Fig. 4B
)
strongly suggest that phosphorylation functions to down-regulate or
inactivate AGT activity, depending on the level of protein
phosphorylation.
Altered Migration of AGT Protein after Phosphorylation and
Dephosphorylation.
Generally, phosphoproteins migrate more slowly relative to their
unphosphorylated counterparts on SDS-polyacrylamide gels. To test
whether this is true for AGT, we reacted recombinant AGT with CK II and
performed Western analysis. The mobility of phosphorylated AGT protein
was slightly retarded compared with the untreated control (Fig. 5A
, compare Lanes 1 and 3). In contrast,
dephosphorylation of the UW228 cell extract resulted in a faster
mobility of the endogenous AGT protein (Fig. 5B
, Lane 2).
Our ongoing studies and other published reports (26
, 27)
have shown the presence of doublet protein bands on the Western blots
of AGT, after SDS-PAGE of human cell extracts; these patterns probably
reflect the existence of differentially phosphorylated forms of the AGT
protein in cells.
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), we used PKA, PKC,
and CK II to phosphorylate purified recombinant AGT protein. All three
kinases phosphorylated the AGT protein in a Mg2+
concentration-dependent manner (Fig. 6A
). No 32P was incorporated in the
absence of Mg2+ with all kinases (Fig. 6A
,
Lane 1), and the rate of phosphorylation increased with increasing
Mg2+ levels. Phosphate incorporation remained
linear for up to 30 min for all kinases. The stoichiometry (mol
phosphate/mol AGT) was 0.15 for PKA, 0.44 for PKC, and 0.28 for CK II.
These results, the means of two separate experiments, demonstrate that
PKC caused efficient phosphorylation of AGT, a finding that came as a
surprise because no strong PKC consensus motifs were detected in the
AGT protein. Because substrate recognition by PKs does not follow
strict patterns (28)
, PKC may have recognized a weak motif
in the NH2-terminal cluster of serine/threonine
phosphorylation sites of the AGT protein (Fig. 1
). A recent study has
also implicated PKC in AGT transcription (29)
. Although
our in vitro studies showed that all well-characterized
serine/threonine kinases, PKA, PKC, and CK II, can use the AGT protein
as a substrate, the question of whether these kinases phosphorylate AGT
under physiological conditions awaits further study.
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), with the degree of inhibition correlating with that of
phosphorylation by the individual kinases. For example, the PKA, which
phosphorylated AGT to the least extent, caused a 15% inhibition
compared with 35 and 55% inhibition by CK II and PKC, respectively.
Prolonged incubation of AGT with the PKs did not result in complete
inhibition of AGT activity. Although the reasons for this are not
clear, it is possible that a partial modification of the protein and/or
the inability of these kinases to phosphorylate the tyrosine may
account for the incomplete inhibition. Taken together, the inhibition
of AGT activity by the purified kinases (Fig. 6B
) and the
data pertaining to the phosphorylation and dephosphorylation of
endogenous AGT (Figs. 4
and 5
) confirm that phosphorylation serves as a
negative regulatory mechanism for AGT.
Conclusions.
Reversible protein phosphorylation catalyzed by the opposing activities
of PKs and phosphatases is a fundamental regulatory mechanism that
controls numerous biological processes including enzyme regulation
(30)
. This article is the first report of the
phosphorylation of human AGT protein. We have provided evidence to
suggest that phosphorylation and dephosphorylation may regulate AGT
activity under physiological conditions. The existence of cellular AGT
as a phosphoprotein under basal conditions is clearly supportive of
this notion. Therefore, a reversible phosphorylation of AGT occurring
in response to various signals may serve as an acute cellular means of
altering the activity levels of this repair protein. Such a regulatory
mode assumes greater significance for AGT, because of its
stoichiometric reaction mechanism and the strict need for de
novo protein synthesis to restore AGT activity after its
inactivation. The circumstances and extent to which AGT is regulated by
phosphorylation in cells is presently unclear; however, the presence of
multiple phosphorylation sites (serine, threonine, and tyrosine) and
consensus motifs for established cellular kinases in the AGT protein
seems to suggest that regulation of this posttranslational modification
is complex and tight.
Tyrosine phosphorylation of AGT is of particular interest because such
a modification is relatively infrequent in cytosolic proteins,
occurring only once in every 100 phosphorylation events
(31)
. Of the three tyrosines at positions 69, 114, and 158
in the human AGT protein, the latter two are located in the putative
DNA binding region and in the vicinity of active site, respectively
(32)
. Tyrosine 114 and tyrosine 158 are highly conserved
in mammalian alkyltransferases, and mutagenesis studies have revealed
their critical contribution to AGT activity (33
, 34)
. Our
motif search (Fig. 1
) indicated that tyrosine 114 is the likely
candidate for phosphorylation in human AGT, and significantly, the same
tyrosine kinase recognition sequence is conserved in all mammalian AGT
proteins. Our studies did not address the contribution of tyrosine
phosphorylation to AGT activity. However, on the basis of the above
considerations, it appears that tyrosine modification may affect AGT
function to a marked extent. Protein phosphorylation may impact other
biological aspects of AGT as well, such as its response to signal
transduction, its nuclear localization, and its protein degradation
(11)
. Also, the levels of kinases and phosphatases in
tumors will determine the phosphorylation status of the AGT protein,
which may in turn account for the variations in AGT activity among
human gliomas and other cancers. Further clarification of this
posttranslational mechanism may provide important clues for elucidating
the cellular regulation of AGT and for devising new therapeutic
approaches targeting this protein, because phosphorylation is
associated with down-regulation of AGT activity.
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FOOTNOTES |
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1 Supported by NIH Grant CA74321 and grants from
the National Childhood Cancer Foundation, Brain Tumor Center, and
Physician Referral Service of the University of Texas M. D. Anderson
Cancer Center (all to K. S. S.). These data were presented in part
(abstract 2658) at the 90th Annual Meeting of the AACR held
April 10–14, 1999 in Philadelphia, PA. 
2 To whom requests for reprints should be
addressed, at Department of Neurosurgery, Box 169, University of Texas
M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX
77030. Phone: (713) 792-3821; Fax: (713) 794-5514; E-mail: ksrivenu{at}mdanderson.org
3 The abbreviations used are: AGT,
O6-alkylguanine-DNA alkyltransferase; BG,
O6-benzylguanine; PK, protein kinase; CK II,
casein kinase II.
Received 10/ 4/99. Accepted 12/ 1/99.
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B
to the ubiquitin-proteasome pathway. Genes Dev., 9: 1586-1597, 1995.This article has been cited by other articles:
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