Identificationofsitesofphosphorylationby G-protein-coupled
receptor kinase2in b-tubulin
Norihiro Yoshida
1,
*, Kazuko Haga
1,2
and Tatsuya Haga
1,2
1
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Japan;
2
Institute for Biomolecular Science,
Faculty of Science, Gakushuin University, Tokyo, Japan
G-protein-coupled receptorkinase2 (GRK2) is known to
specifically phosphorylate the agonist-bound forms of
G-protein-coupled receptors (GPCRs). This strict specificity
is due at least partly to activation of GRK2 by agonist-
bound GPCRs, in which basic residues in intracellular
regions adjacent to transmembrane segments are thought to
be involved. Tubulin was found to be phosphorylated by
GRK2, but it remains unknown if tubulin can also serve as
both a substrate and an activator for GRK2. Purified
tubulin, phosphorylated by GRK2, was subjected to
biochemical analysis, and the phosphorylationsites in
b-tubulin were determined to be Thr409 and Ser420. In
addition, the Ser444 in b
III
-tubulin was also indicated to be
phosphorylated by GRK2. The phosphorylationsites in
tubulin for GRK2 reside in the C-terminal domain of
b-tubulin, which is on the outer surface of microtubules.
Pretreatment of tubulin with protein phosphatase type-2A
(PP2A) resulted in a twofold increase in the phosphorylation
of tubulin by GRK2. These results suggest that tubulin is
phosphorylated in situ probably by GRK2 and that the
phosphorylation may affect the interaction of microtubules
with microtubule-associated proteins. A GST fusion protein
of a C-terminal region of b
I
-tubulin (393–445 residues),
containing 19 acidic residues but only one basic residue, was
found to be a good substrate for GRK2, like full-length
b-tubulin. These results, together with the finding that
GRK2 may phosphorylate synuclein and phosducin in their
acidic domains, indicate that some proteins with very acidic
regions but without basic activation domains could serve as
substrates for GRK2.
Keywords: G-protein-coupledreceptor kinase; protein
phosphorylation; tubulin.
Many G-protein-coupled receptors (GPCRs) including
rhodopsin, muscarinic acetylcholine receptors, and b-adre-
nergic receptors are known to be phosphorylated in a light-
dependent or agonist-dependent manner by members of the
protein kinase family called G-protein-coupled receptor
kinases (GRKs) [1]. GRKs constitute a subgroup of the
serine/threonine kinase superfamily and are characterized
by their strict substrate specificity, i.e. they only recognize
the stimulated forms of GPCRs. The phosphorylation sites
in rhodopsin [2] and b
2
-adrenergic receptors [3] for GRK1
and GRK2, respectively, are located in their C-termini, and
those in muscarinic acetylcholine receptor M
2
subtypes (M
2
receptors) [4], M
3
receptors [5], and a
2
-adrenergic receptors
[6] for GRK2 are in the central parts of their third
intracellular loops. No strict consensus sequence for GRK-
mediated phosphorylation has been found among these
phosphorylation sites, except that acidic amino-acid resi-
dues near the phosphorylationsites may be required [7].
Peptides corresponding to these phosphorylationsites are
generally poor substrates for GRK1 or GRK2, but their
phosphorylation is greatly stimulated by rhodopsin [8],
b
2
-adrenergic receptors [9] or M
2
receptors [10] in a light-
dependent or agonist-dependent manner. GRK2, but not
GRK1, is also stimulated by G-protein bc subunits, and this
phosphorylation is synergistically stimulated by agonist-
bound receptors and G-protein bc subunits [11–14]. These
results indicate that light-exposed rhodopsin, agonist-bound
b-adrenergic receptors, or M
2
receptors function both as
substrates and activators, and explain, at least partly, why
the substrates of GRK2 are restricted to agonist-bound
receptors in spite of the absence of a strict consensus
sequence among various phosphorylation sites. As phos-
phorylation site-deleted rhodopsin [15] and M
2
receptors
[10] also act as activators of GRK2, the activation sites are
thought to be different from the phosphorylation sites.
Possible activation sitesin M
2
receptors are suggested to be
several portions of intracellular loops adjacent to trans-
membrane segments, because the peptides corresponding to
these regions stimulated phosphorylationof synthetic
peptides corresponding to the phosphorylationsitesin M
2
receptors [14]. These regions are assumed to undergo a
conformational change on agonist binding and to be
involved in the interaction with G-proteins [16,17]. Further-
more, mastoparan, which is known to mimic agonist-bound
Correspondence to T. Haga, Institute for Biomolecular Science,
Faculty of Science, Gakushuin University, Mejiro 1-5-1,
Toshima-ku, Tokyo 171-8588, Japan.
Fax: + 81 35992 1034, Tel.: + 81 35992 1033,
E-mail: tatsuya.haga@gakushuin.ac.jp
Abbreviations: GPCR, G-protein-coupled receptor; GRK, G-protein-
coupled receptor kinase; PP2A, phosphatase 2A; MAP,
microtubule-associated protein; PVDF, poly(vinylidene difluoride);
GST, glutathione S-transferase.
*Present address: Otsuka Pharmaceutical Co. Ltd, Research Institute
for Pharmacological and Therapeutical Development,
Tokushima, Japan.
(Received 23 October 2002, revised 7 January 2003,
accepted 17 January 2003)
Eur. J. Biochem. 270, 1154–1163 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03465.x
receptors and activates G-proteins [18], has been shown to
stimulate GRK1 [15] and GRK2, particularly in the
presence of G-protein bc subunits [14]. All these peptides
with GRK2-stimulating activity, including mastoparan, are
basic peptides.
Recent studies have suggested that GRK may phos-
phorylate substrates other than the stimulated forms of
GPCRs. Tubulin is the first nonreceptor protein found to be
phosphorylated by GRK2 and GRK5, although its phos-
phorylation sites have not been identified yet [19–21]. Other
nonreceptor substrates for GRK2 have been reported,
including synucleins [22], phosducin, and phosducin-like
protein [23]. The phosphorylationsitesin synucleins and
phosducin are located in their C-terminal domains, which
include many acidic residues but few basic residues. It
remains unknown, however, whether the C-terminal pep-
tides serve as good substrates for GRK2 by themselves or
do so only in the presence of activating domains in another
part of these proteins. We have attempted to identify
phosphorylation sites for GRK2 in tubulin as a first step to
determining if tubulin serves as both a substrate and an
activator for GRK2, as was shown in the case of stimulated
forms of GPCRs.
Here, we show that tubulin is phosphorylated by GRK2
in a very acidic C-terminal domain and that the C-terminal
peptide of tubulin is a good substrate for GRK2, suggesting
that the presence of a basic activation domain is not
necessary for the protein to be a substrate for GRK2. In
addition, we present evidence that tubulin is phosphorylated
in situ at the sites phosphorylated by GRK2.
Materials and methods
Materials
Phenyl-sepharose, heparin-sepharose, glutathione-seph-
arose 4B, sephadex G-50 fine, [c-
32
P]ATP, the pGEX4T-3
vector, and an ECL chemiluminescence detection system
were purchased from Amersham Pharmacia Biotech.
Achromobacter protease I and endoproteinase Asp-N were
purchased from Wako Pure Chemical Industries. KOD
polymerase, Pfu turbo polymerase, the pBluescript vector,
and restriction enzymes were from Toyobo. C
18
RP-HPLC
and DEAE-5PW columns were from Tosoh. A thermo
sequence fluorescent-labeled primer cycle sequencing kit
was purchased from Perkin–Elmer. TLC plates were
purchased from Merck, and human erythrocyte phospha-
tase 2A (PP2A) was from Upstate Biotechnology Inc. Other
reagents used were of the highest grade commercially
available.
Protein expression and purification
GRK2 was overexpressed in and purified from Sf9 insect
cells as described previously with some modifications [10].
The infected Sf9 cells were homogenized in 20 m
M
Hepes/
KOH (pH 7.0), containing 2 m
M
MgCl
2
,1m
M
dithiothre-
itol, and 0.5 m
M
phenylmethanesulfonyl fluoride (solution
A; 20 mL per cell pellet from 1 L culture). The homogenate
was centrifuged, and then the pellet was homogenized in
solution A supplemented with 0.5
M
KCl. Most of the
GRK2 activity was recovered in the supernatant obtained
by centrifugation at 42 000 g for 20 min. Ammonium
sulfate was added to the extract to a saturation level of 20%.
After centrifugation, a saturated ammonium sulfate solu-
tion was added to the supernatant to give a final concen-
tration of 30%, and then the suspension was centrifuged
and the resulting pellet dissolved in solution A (15 mL).
This solution was applied to a phenyl-Sepharose column
(5 mL) equilibrated with 20 m
M
Hepes/KOH (pH 7.0),
containing 1 m
M
dithiothreitol, 0.5 m
M
phenylmethane-
sulfonyl fluoride, and 1
M
ammonium sulfate at a flow rate
of 1 mLÆmin
)1
. After the column had been washed
thoroughly, proteins were eluted with a linear gradient of
1.0–0
M
ammonium sulfate (1.0 and 0
M
, 20 mL each) and
collected in fractions of2 mL each. The fractions containing
tubulin-phosphorylating activity were combined, dialyzed
against 20 m
M
Hepes/KOH (pH 7.0)/50 m
M
NaCl, and
then applied to a heparin column (1 mL) equilibrated with
the dialysis buffer. Proteins were eluted with a linear
gradient of 50–500 m
M
NaCl in 20 m
M
Hepes/KOH
(pH 7.0) (30 mL in total) and collected in fractions of
1.5 mL each. Each fraction was assayed for GRK2, and
subjected to SDS/PAGE on a 12% acrylamide gel. The
purified GRK2 was mixed with an equal volume of glycerol
as a stabilizer and stored at )80 °C until use. Crude tubulin,
which contained microtubule-associated proteins (MAPs),
was prepared from porcine brains by the polymerization–
depolymerization procedure, which was performed three
times, as described previously [24]. The tubulin was further
purified by phosphocellulose chromatography [25].
Phosphorylation of tubulin
Tubulin was phosphorylated with GRK2 as described
previously with some modifications [19]. Briefly, various
concentrations of tubulin were incubated with 40 n
M
GRK2 in a buffer comprising 50 l
M
[c-
32
P]ATP
(100 c.p.m.Æpmol
)1
), 20 m
M
Tris/HCl(pH7.4),50m
M
KCl, 2 m
M
EDTA, 0.5 m
M
EGTA, and 5 m
M
MgCl
2
at
30 °C, followed by SDS/PAGE. Incorporation of radio-
activity into the tubulin was visualized by autoradiography
and quantified with a Fuji BioImage BAS2000 analyzer.
Overlaying and detection of GRK2
Purified tubulin was reduced and carboxymethylated
essentially as described previously [26]. Purified tubulin
was lyophilized and then dissolved in 140 lL6
M
guanidi-
nium hydrochloride in 0.1
M
Tris/HCl (pH 8.5), 40 lL
propan-2-ol, and 2 lL 2-mercaptoethanol by incubation at
room temperature for 2 h. The tubulin solution was then
carboxymethylated by mixing it with 1 lL1
M
iodoacetic
acid in 1
M
NaOH, followed by incubation of the mixture at
room temperature in the dark for 40 min. The reaction was
terminated by the addition of excess 2-mercaptoethanol,
and then the mixture was passed through a column of
Sephadex G-50 fine (2 mL) previously equilibrated with
50 m
M
ammonium carbonate (pH 9.0). The carboxymethy-
lated tubulin was subjected to SDS/PAGE and then
transferred to a poly(vinylidene difluoride) (PVDF) mem-
brane [27,28]. The PVDF membrane was incubated in
blocking buffer [0.1% (v/v) Tween 20 and 5% (w/v) nonfat
dry milk in NaCl/P
i
]for1hat4°C, and subsequently
Ó FEBS 2003 Sitesofphosphorylationby GRK2 in tubulin (Eur. J. Biochem. 270) 1155
washed three times with binding buffer [0.1% (v/v)
Tween 20 and 0.5% (w/v) nonfat dry milk in NaCl/P
i
].
ThePVDFmembranewasthenincubatedwithGRK2in
binding buffer overnight at 4 °C. After the PVDF mem-
brane had been washed three times with binding buffer,
GRK2 was detected by incubating the PVDF membrane
with anti-GRK2 IgG. For immunological detection, horse-
radish peroxidase-conjugated anti-IgG antibodies and an
ECL chemiluminescence system were used according to the
manufacturer’s instructions.
Digestion of tubulin
Phosphorylated and then carboxymethylated tubulin
(100 lg) was treated with 1 lg Achromobacter protease I
(EC 3.4.21.50) in 200 lL 100 m
M
ammonium carbonate
buffer (pH 9.0) at 37 °C for 60 min. The digested peptides
were applied to a DEAE-5PW column equilibrated with
50 m
M
ammonium carbonate (pH 9.0)/50 m
M
NaCl at a
flow rate of 0.5 mLÆmin
)1
. After the column had been
washed, the peptides were eluted with a linear gradient of
50–500 m
M
NaCl in 50 m
M
ammonium carbonate, pH 9.0
(30 mL in total) and collected in fractions of 1 mL each.
Radioactivity was detected by Cerenkov counting. The
fractions containing the phosphopeptides were combined
and digested overnight with 10 lg endoproteinase Asp-N at
30 °C. The reaction product was applied directly to a C
18
RP-HPLC column, which was eluted with a linear gradient
of 0–50% acetonitrile containing 0.1% trifluoroacetic acid
in 50 min at a flow rate of 0.3 mLÆmin
)1
. The amino-acid
sequences of the radioactive peptides were determined with
a Hewlett–Packard G1000A Protein Sequencer.
Phosphoamino-acid analysis by TLC
A portion of the radioactive peptides eluted from the C
18
column was lyophilized, resuspended in 6
M
HCl, and then
hydrolyzed by incubation at 110 °C for 60 min. The
hydrolysate was lyophilized and then subjected to TLC
with pyridine/acetic acid/water (1 : 10 : 189, v/v). The
radioactive phosphoamino acids were visualized by auto-
radiography. The TLC plate was sprayed with 0.7%
ninhydrin in acetone and heated in an oven at 65 °C to
visualize the standard phosphoamino acids.
Cloning ofb-tubulin and mutagenesis of its
phosphorylation sites
Poly(A)-rich RNA was prepared from rat and mouse brains
with Moloney murine leukemia virus reverse transcriptase
(Toyobo) and then used to construct a cDNA library. The
DNA fragment encoding the full-length rat b
I
-tubulin
(accession No. AB011679) or mouse b
III
-tubulin (accession
no. NM_023279) was amplified by PCR using the rat or
mouse brain cDNA library as a template. The PCR
products were digested with EcoRI–NotI and then cloned
into plasmid vector pBluescript II KS(–). For construction
of mutant b
I
-tubulin and b
III
-tubulin, Thr409 and Ser420 of
b
I
-tubulin and Thr409, Ser420 and Ser444 of b
III
-tubulin
were replaced with Ala using the inverted amplification
method [29]. Oligonucleotide primers were designed in
inverted tail-to-tail directions to amplify the cloning vector
together with the inserts. PCR was performed with Pfu
turbo polymerase. cDNAs encoding the wild-type and
mutant b-tubulins were excised as EcoRI–NotI fragments
and then subcloned into EcoRI–NotI-digested expression
vector pGEX4T-3, followed by transformation into
Escherichia coli and expression as fusion proteins with
glutathione S-transferase (GST; GST-b-tubulins). A fusion
protein with GST of a peptide corresponding to positions
393–445 of rat b
I
-tubulin was also expressed in E. coli using
an expression vector, pGEX4T-3 (GST-b-tubulinC). These
GST fusion proteins were purified using glutathione-Seph-
arose by the procedure recommended by the manufacturer,
as described previously [10].
Dephosphorylation of tubulin
Tubulin purified from porcine brains was subjected to
dephosphorylation with PP2A (0.2 U) at 30 °C for 60 min.
The dephosphorylation buffer contained 50 m
M
Mes
(pH 6.8), 0.1 m
M
EDTA, 1 m
M
EGTA, 5 m
M
MgCl
2
,
0.2 mgÆmL
)1
BSA, and 1 m
M
2-mercaptoethanol. The
dephosphorylation reaction was terminated by adding
10 n
M
okadaic acid, followed byphosphorylationof the
dephosphorylated tubulin by GRK2 at 30 °C as above. In
the control sample, PP2A was incubated with 10 n
M
okadaic acid before the addition of tubulin. To follow the
time course of dephosphorylation, tubulin was first phos-
phorylated in the presence of [c-
32
P]ATPbyGRK2and
then subjected to dephosphorylation by PP2A, followed by
SDS/PAGE and quantification of the radioactivity remain-
ing in the tubulin.
Results
GRK2 binds specifically to b-tubulin
The a and b isotypes of tubulin were separated from each
other by carboxymethylation and subsequent SDS/PAGE.
After electrophoresis and Western blotting, the PVDF
membrane was incubated with a purified preparation of
GRK2 and then GRK2 antibodies. As shown in Fig. 1,
GRK2 was found to interact only with b-tubulin. This is
consistent with the study by Carman et al.[21],inwhich
GRK2 phosphorylated b-tubulin but not a-tubulin. There-
fore, these results indicate that GRK2 binds to and
phosphorylates b-tubulin specifically.
Partial digestion of phosphorylated tubulin
Tubulin phosphorylated by GRK2 was cleaved on the
C-terminal side of Lys with Achromobacter protease I and
on the C-terminal sides of Lys and Arg with trypsin. The
cleaved peptides were subjected to analysis by SDS/PAGE
with 18% acrylamide in Tricine. The smallest fragment
obtained on treatment with Achromobacter protease I or
trypsin had an apparent molecular mass of 6 kDa (data not
shown). We examined the amino-acid sequence of
b-tubulin, looking for the region with the expected length
after the Achromobacter protease I or trypsin treatment, and
found that the C-terminus of the b-tubulin had the most
likely sequence to be phosphorylated by GRK2. The
sequence between 392 and 430 is the same in the b
II
and
1156 N. Yoshida et al.(Eur. J. Biochem. 270) Ó FEBS 2003
b
III
tubulin isotypes of pig, and b
I
, b
II
, b
III
,andb
IV
isotypes
of mouse (Table 1). The sequence between residue 431 and
the C-terminus differs from one isotype to another, but
there are no Ser or Thr residues in the region except for
Ser444 in b
III
-tubulin [30]. Neither Lys nor Arg is present
between Lys392 and the C-terminus except for Lys450 in
b
III
-tubulin. Thus, the fragment obtained by treatment with
Achromobacter protease I or trypsin is expected to have
53–58 residues, which corresponds to the phosphorylated
6-kDa band shown by SDS/PAGE. This C-terminal region
of b-tubulin from Ala393 to Lys450 is extremely acidic with
20 acidic residues and only two basic His396 and Lys450
residues, and it has three Ser and three Thr residues.
Separation of radiolabeled peptides and analysis
of phosphoamino acids
Phosphorylated tubulin was carboxymethylated and then
digested with Achromobacter protease I. The digested sample
was loaded on to a DEAE column. All the radioactivity
Table 1. Sites and potential sites for GRK2-mediated phosphorylation. Phosphorylationsites for GRK2 were identified for rhodopsin [2],
b
2
-adrenergic receptors [3], and a-synuclein and b-synuclein [22]. Potential phosphorylationsites for GRK2 are indicated for a
2A
-receptors [39], M
2
receptors [36], M
3
receptors [5], phosducin and phosducin-like protein [23]. The phosphorylationsites and potential sites are indicated in bold type
as S or T. Acidic and basic amino acids are denoted by italics and underlining, respectively.
Protein Sequence
Rhodopsin (bovine, 309–348, C-terminal)
MNKQFRNCMLTTICCGKNPLGDDEASATVSKTETSQVAPA-OH
b
2
-Adrenoceptor
(human, 362–413, C-terminal)
EQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL-OH
a
2A
-Adrenoceptor (human, 279–323,
third intracellular loop)
EPAPAGPRDTDALDLEESSSSDHAERPPGPRRPERGPRGKGKARA
M
2
(human, 278–321, third intracellular loop) EEKESSNDSTSVSAVASNMRDDEITQDENTVSTSLGHSKDENSK
M
3
(rat, 317-361, third intracellular loop) KSWKPSAEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGS ETR
b
III
-Tubulin (mouse, 392–450, C-terminal) KAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATADEEGEMY
EDDDEE
SEAQGPK-OH
a-Synuclein (human, 103–140, C-terminal) NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA-OH
b-Synuclein (human, 95–134, C-terminal) PEEVAQEAAEEPLIEPLMEPEGESYEDPPQEEYQEYEPEA-OH
Phosducin (rat, 206–246, C-terminal) EQFAEEFFAADVESFLNEYGLLPEREIHDLGQTNTEDEDIE-OH
Phosducin-like protein
(rat, 279–301, C-terminal)
VLVLTSVRNSATCHSEDSDLEID-OH
Fig. 1. b-Tubulin binds to GRK2. Purified
tubulin and carboxymethylated tubulin
(CM-tubulin) were subjected to SDS/PAGE
and then stained with Coomassie Brilliant
Blue or transferred to PVDF membranes. The
PVDF membranes were incubated with puri-
fied GRK2 (0.6 lgÆmL
)1
) and then with
GRK2 antibodies, as described in Materials
and methods.
Ó FEBS 2003 Sitesofphosphorylationby GRK2 in tubulin (Eur. J. Biochem. 270) 1157
bound to the column, none being detected in the flow-
through fraction. As shown in Fig. 2A, most of the
radioactivity was eluted as a single peak. The N-terminal
sequence determined for the 6-kDa fragment was
AFLHWYTGEG-, which was identical with the sequence
of residues 393–402 in the C-terminus of porcine b-tubulins
[30].
The 6-kDa fragment was further digested with endopro-
teinase Asp-N and then subjected to RP-HPLC on a C
18
column. Phosphopeptides were eluted at 20% acetonitrile
as two peaks with a linear gradient of 0–50% acetonitrile
(Fig. 2B). The phosphopeptides obtained were analyzed by
two-dimensional mapping on TLC plates. Each of the two
peak fractions from the C
18
column gave a single spot on
TLC mapping (data not shown). Edman sequence analysis
of each peptide (peptide 1 and peptide 2) revealed that
peptide 1 had the sequence DEMEFTEAESNMN(404–
416), and peptide 2 had the sequence DLVSEYQQYQ(417–
426). Acid hydrolysis followed by TLC analysis revealed
only labeled phosphothreonine on peptide 1 and labeled
phosphoserine on peptide 2 (Fig. 3). These results indicate
that the phosphorylationsites for GRK2 are Thr409 and
Ser420, but not Ser413.
Phosphorylation by GRK2 of GST fusion proteins
of full-length b
I
-tubulin, b
III
-tubulin, and C-terminal
peptides of b
I
-tubulin expressed in
E. coli
We cloned the b
I
-tubulin and b
III
-tubulin genes. GST fusion
proteins of b
I
-tubulin (GST-b
I
-tubulin), its C-terminal
peptide (393–445) (GST-b
I
-tubulinC), and b
III
-tubulin
(GST-b
III
-tubulin) were expressed in E. coli and then
subjected to phosphorylationby GRK2 with different
substrate concentrations (Fig. 4). Each substrate was
found to be phosphorylated by GRK2 at similar rates.
The K
m
values for GST-b
I
-tubulin, GST-b
III
-tubulin and
GST-b
I
-tubulinC were estimated to be 2.6, 6 and 12 l
M
,
respectively. The K
m
values for GST-b
I
-tubulin and
Fig. 2. Elution profile of
32
P-labeled peptides on DEAE and C
18
RP-HPLC columns. (A) Phosphorylated tubulin was carboxymethyl-
ated, digested with Achromobacter protease I at 37 °C for 30 min, and
then applied to a DEAE column (0.75 · 7.5 cm). The elution of
peptides and radioactivity was monitored by measuring A
280
(upper
line) and Cerenkov radiation (lower line), respectively. Edman degra-
dation of the fraction, which included the major phosphopeptide,
revealed the N-terminal sequence to be AFLHWYTGEG(393–402).
(B) The pooled fractions from the DEAE column were digested
overnight with endoproteinase Asp-N at 30 °Candthenappliedtoa
C
18
RP-HPLC column (0.46 · 25 cm). Elution was monitored by UV
absorption at 214 nm (upper line), and radioactivity was measured by
Cerenkov counting (lower line). Two phosphopeptides were eluted
from the C
18
RP-HPLC column, which were subjected to Edman
degradation and sequence determination. The sequence of peptide 1
was determined to be DEMEFTEAESNMN(404–416) and that of
peptide 2 to be DLVSEYQQYQ(417–426).
Fig. 3. [
32
P]Phosphoamino-acid analysis on TLC plates. [
32
P]Phospho-
peptides eluted from a DEAE or C
18
RP-HPLC column (peptide 1 and
peptide 2) were partially hydrolyzed with HCl and then analyzed by
TLC. Autoradiograms of the TLC plates are shown, together with
standard phosphoamino acids.
1158 N. Yoshida et al.(Eur. J. Biochem. 270) Ó FEBS 2003
GST-b
III
-tubulin are comparable to those reported for the
phosphorylation of tubulin purified from porcine brains
(0.4–3 l
M
) [19–21].
Phosphorylation by GRK2 of GST fusion proteins
of b-tubulin mutants
To confirm that Thr409 and Ser420 are the only phos-
phorylation sites for b-tubulin, we constructed mutants of
b
I
-tubulin with Ala409 and/or Ala420 in place of Thr409
and Ser420. In addition, we constructed mutants of b
III
-
tubulin with Ala409, Ala420, and Ala444 or Ser444 to
examine whether Ser444 in b
III
-tubulin is phosphorylated by
GRK2. These mutant forms were expressed in E. coli as
GST fusion proteins and then analyzed with respect to their
phosphorylation by GRK2. As demonstrated in Fig. 5A,
compared with the wild-type b
I
-tubulin (GST-b
I
-tubulin),
the mutant b
I
-tubulin (T409A and S420A) was less than
50% phosphorylated by GRK2 and the double mutant
b
I
-tubulin (T409A/S420A) was hardly phosphorylated at
all. These results confirm that Thr409 and Ser420 are the
only residues in b
I
-tubulin phosphorylated by GRK2. On
the other hand, compared with wild-type b
III
-tubulin (GST-
b
III
-tubulin), the double mutant b
III
-tubulin (T409A/
S420A) was 30% phosphorylated, and the triple mutant
b
III
-tubulin (T409A/S420A/S444A) was hardly phosphory-
lated at all (Fig. 5B). This result indicates that Ser444 of
b
III
-tubulin is also a site of phosphorylation.
Phosphorylation of phosphatase-treated tubulin
Tubulin purified from porcine brains was phosphorylated
with GRK2 and then dephosphorylated with PP2A. About
80% of the phosphate was removed from the tubulin on
treatment with 0.2 U PP2A for 40 min at 30 °C,aswasthe
case for b
III
-tubulin [31,32] (Fig. 6). We treated the purified
tubulin with PP2A and then phosphorylated it with GRK2.
As shown in Fig. 7, the amount ofphosphorylation doubled
on pretreatment with PP2A. This result indicates that
tubulin had been phosphorylated when purified and that the
endogenous phosphorylation is susceptible to PP2A and
that the site can be phosphorylated by GRK2.
Discussion
In this report, we have shown that the phosphorylation sites
in tubulin for GRK2 reside in the C-terminal domain of
b-tubulin, and that two (Thr409 and Ser420) of five Ser or
Thr residues in this domain are phosphorylated. As the four
isotypes of b-tubulin, b
I
, b
II
, b
III
and b
IV
,havethesame
sequence around the phosphorylation sites, it is most likely
that all these isotypes serve as substrates for GRK2.
The extent ofphosphorylationby GRK2 was found to be
increasedwhentubulinwaspretreatedwithPP2Aafterits
purification from porcine brain. This result indicates that
tubulin is phosphorylated in situ at sites from which
phosphate may be removed by PP2A and to which
phosphate may be added by GRK2. One of the most likely
candidate sites is Ser444 in b
III
-tubulin, although it is also
possible that Thr409, Ser420, and other residues are the
relevant sites. Evidence for this is that Ser444 has been
identified as the phosphorylation site in brain-specific b
III
-
tubulin phosphorylated in cultured cells [33] and in the brain
[34]. Furthermore, Khan and coworkers have reported that
phosphate on the Ser444 residue of b
III
-tubulin is resistant
to a wide variety of phosphatases, except human erythrocyte
Fig. 4. Phosphorylationby GRK2 of GST fusion proteins of b
I
-tubulin and b
III
-tubulin (GST-b-tubulin) and the C-terminal peptide of b
I
-tubulin (GST-
b
I
-tubulinC). The indicated concentrations of GST fusion proteins were subjected to phosphorylation with GRK2 in the presence of 50 l
M
[c-
32
P]ATP and 40 n
M
GRK2 for 10 min, followed by SDS/PAGE, and radioactivity counting of the tubulin band. Molar concentrations of fusion
proteins were calculated from the molecular mass: GST, 27.5 kDa; GST-b
I
-tubulin and GST-b
III
-tubulin,82.5kDa;GST-b
I
-tubulinC, 33.5 kDa.
CurveswerefittedtotheMichaelis–Mentenequation,andK
m
values were estimated to be 2.5 l
M
(GST-b
I
-tubulin), 6 l
M
(GST-b
III
-tubulin) and
12 l
M
(GST-b
I
-tubulinC). These experiments were repeated three times with essentially the same results.
Ó FEBS 2003 Sitesofphosphorylationby GRK2 in tubulin (Eur. J. Biochem. 270) 1159
PP2A [31], which is known to bind to polymerized tubulin
[32]. Moreover, we demonstrated phosphorylation of
Ser444 by GRK2 using recombinant tubulin mutants,
although the phosphorylation was not detected for tubulin
purified from porcine brain. These results suggest that
GRK2 is the kinase that phosphorylates Ser444, although
we cannot exclude the involvement of other kinases such as
casein kinase II [35]. This assumption is supported by the
observation that GRK2 is localized with microtubules in
intact cells and the localization is facilitated by agonist-
bound GPCRs [20].
The phosphorylationsites for GRK2 have been deter-
mined for rhodopsin [2], b
2
-adrenergic receptors [3], and
synucleins [22]. Serine and threonine clusters have also been
shown to be phosphorylationsites for GRK2 in M
2
receptors [36], M
3
receptors [5], a
2A
-adrenergic receptors
[37], and phosducin [23], although the phosphorylated
amino-acid residues have not been determined definitely.
These phosphorylationsites and phosphorylation site
candidates are shown in Table 1. Each of these phosphory-
lation sites resides in an acidic domain with a fairly long
span. It may be a prerequisite for phosphorylation by
GRK2 that the phosphorylationsites are in an acidic
domain. However, it is not the only condition for
phosphorylation that the Ser and Thr residues are in the
acidic domain, as GRK2 phosphorylated Thr409, Ser420
and Ser444 but not Thr399, Ser413, and Thr429 in the
C-terminal domain of b
III
-tubulin. Further research is
necessary into what discriminates phosphorylated from
nonphosphorylated residues.
Initially, we hypothesized that tubulin may serve as both
a substrate and an activator for GRK2 and that it contains
a basic GRK2-activating domain besides a substrate
domain. This working hypothesis is not supported by the
present findings that the C-terminal peptide of b
I
-tubulin
(b
I
-tubulinC), which is very acidic and does not contain a
basic domain, is as good a substrate as full-length tubulin.
Even if tubulin contains a basic GRK2-activating domain,
the effect of the putative domain should not be important
because the K
m
values for b
I
-tubulin and b
I
-tubulinC only
differ by a factor of 5. Therefore, it is likely that tubulin is a
substrate for GRK2 for a different reason from that in the
case of agonist-bound GPCRs.
Synucleins and phosducin have been reported to be
substrates for GRK2, but it is not known if they have basic
domains which serve as activators for GRK2. However, we
have noticed a common characteristic of tubulin, synucleins,
and phosducin, i.e. all three proteins have very acidic
C-terminal domains that include phosphorylation sites. The
C-terminal domain of b
III
-tubulin contains 20 acidic
residues in a span of 58 residues (35%) with only two basic
residue (His and Lys). The C-terminal domains of synuc-
leins (a and b) also contain phosphorylationsitesin very
acidic domains, with 37–40% of acidic residues and no basic
residues (Table 1). The phosphorylationsitesin GPCRs are
also in an acidic domain, but the acidic nature is much less
Fig. 5. Phosphorylationof GST-b
I
-tubulin and
b
III
-tubulin mutants. (A) Residues Thr409 and
Ser420, and both Thr409 and Ser420 residues
in b
I
-tubulin were replaced with alanine resi-
dues, yielding mutants T409A, S420A, and
T409A/S420A, respectively. (B) Residues
Thr409 and Ser420 and/or Ser444 residues
were replaced with alanine residues, yielding
mutants T409A/S420A and T409A/S420A/
S444A. GST fusion proteins of these mutants
were expressed in E. coli and then purified as
described in Materials and methods. These
b-tubulin mutants were subjected to phos-
phorylation with GRK2. The values are the
means of three independent experiments for
each b
I
-tubulin and b
III
-tubulin mutants with
similar results and are expressed as percent-
ages of the control value for wild-type GST-b
I
or b
III
-tubulin. Error bars represent means
±SD.
1160 N. Yoshida et al.(Eur. J. Biochem. 270) Ó FEBS 2003
evident. The presence of very acidic domains, particularly in
the C-termini of nonreceptor substrates, may constitute a
criterion for phosphorylationby GRK2.
The C-terminal domain containing Thr409 and Ser420
has been shown to form an a-helix (H12, 408–423 residues)
and to be located on the outermost surface of microtubules
[38]. The C-terminal residues including Ser444 of b-tubulin,
which are lacking in the structure model, are also thought to
be located on the outermost surface of microtubules. This is
consistent with the findings that microtubules as well as
tubulin dimer can be phosphorylated by GRK2 and that
phosphorylated tubulin can polymerize into microtubules
[19]. The C-termini of a and b tubulin are thought to
be involved in the binding of MAPs and motor proteins
[39,40]. MAPs and motor proteins are known to have major
roles in microtubule assembly, organelle transport,
and mitosis. It is possible that phosphorylationof the
C-terminus ofb-tubulinby GRK2 affects the microtubule
dynamics or cellular mechanisms by affecting the binding of
MAPs or motor proteins. In addition, a series of recent
studies have demonstrated that Ga or Gbc subunits interact
directly with tubulin [41–43] and that muscarinic receptor
activation induces transient translocation of tubulin to the
plasma membrane [44]. Furthermore, microtubules have
been suggested to mediate the internalization of b-adrener-
gic receptors [45]. The GRK-mediated phosphorylation of
tubulin may affect physiological processes including
GPCRs, and the interaction of GRK2 with tubulin may
have an effect on the function of GRK2.
Acknowledgements
We thank Professor K. Matsushima and Mr Y. Terashima for their
help in determining the peptide sequences. This work was supported in
part by grants from the Japan Society for the Promotion of Science
(Research for Future Program), and from the Japan Science and
Technology Corporation (CREST).
References
1. Pitcher, J.A., Freedman, N.J. & Lefkowitz, R.J. (1998) G protein-
coupled receptor kinases. Annu.Rev.Biochem.67, 653–692.
2. Ohguro, H., Rudnicka-Nawrot, M., Buczylko, J., Zhao, X.,
Taylor, J.A., Walsh, K.A. & Palczewski, K. (1996) Structural and
enzymatic aspects of rhodopsin phosphorylation. J. Biol. Chem.
271, 5215–5224.
Fig. 7. Effects of PP2A treatment on phosphorylationof tubulin by
GRK2. Purified tubulin was incubated with or without PP2A for
60 min at 30 °C followed byphosphorylationby GRK2 at 30 °C for
30 min. In samples designated PP2A + okadaic acid, PP2A was
preincubated with okadaic acid before the phosphorylation reaction.
The indicated values are means of triplicate determinations in one of
three independent experiments with similar results. Error bars repre-
sent means ± SD.
Fig. 6. Dephosphorylation by PP2A of phosphorylated tubulin. Tubulin
purified from porcine brains was phosphorylated with [c-
32
P]ATP by
GRK2 at 30 °C for 30 min as described above and then incubated with
or without PP2A (0.2 U) for the indicated time at 30 °C, followed by
SDS/PAGE and counting of radioactivity on tubulin band. The
indicated values are means of triplicate determinations in one of three
independent experiments with similar results and are expressed as
percentages of the amount of tubulin phosphorylated before addition
of PP2A. Error bars represent means ± SD.
Ó FEBS 2003 Sitesofphosphorylationby GRK2 in tubulin (Eur. J. Biochem. 270) 1161
3. Fredericks, Z.L., Pitcher, J.A. & Lefkowitz, R.J. (1996) Identifi-
cation of the G protein-coupled receptorkinase phosphorylation
sites in the human b
2
-adrenergic receptor. J. Biol. Chem. 271,
13796–13803.
4. Nakata, H., Kameyama, K., Haga, K. & Haga, T. (1994) Loca-
tion of agonist-dependent-phosphorylation sitesin the third
intracellular loop of muscarinic acetylcholine receptors (m2
subtype). Eur. J. Biochem. 220, 29–36.
5. Wu, G., Bogatkevich, G.S., Mukhin, Y.V., Benovic, J.L., Hilde-
brandt, J.D. & Lanier, S.M. (2000) Identificationof G
bc
binding
sites in the third intracellular loop of the M(3)-muscarinic receptor
and their role inreceptor regulation. J. Biol. Chem. 275, 9026–
9034.
6. Liggett, S.B., Ostrowski, J., Chesnut, L.C., Kurose, H., Raymond,
J.R., Caron, M.G. & Lefkowitz, R.J. (1992) Sitesin the third
intracellular loop of the a
2A
-adrenergic receptor confer short term
agonist-promoted desensitization. J. Biol. Chem. 267, 4740–4746.
7. Onorato, J.J., Palczewski, K., Regan, J.W., Caron, M.G., Lef-
kowitz, R.J. & Benovic, J.L. (1991) Role of acidic amino acids in
peptide substrates of the b-adrenergic receptorkinase and rho-
dopsin kinase. Biochemistry 30, 5118–5125.
8. Fowles, C., Sharma, R. & Akhtar, M. (1988) Mechanistic studies
on the phosphorylationof photoexcited rhodopsin. FEBS Lett.
238, 56–60.
9. Chen, C.Y., Dion, S.B., Kim, C.M. & Benovic, J.L. (1993) Beta-
adrenergic receptor kinase. Agonist-dependent receptor binding
promotes kinase activation. J. Biol. Chem. 268, 7825–7831.
10. Kameyama, K., Haga, K., Haga, T., Moro, O. & Sadee, W. (1994)
Activation of a GTP-binding protein and a GTP-binding-protein-
coupled receptorkinase (b-adrenergic-receptor kinase-1) by a
muscarinic receptor m2 mutant lacking phosphorylation sites.
Eur. J. Biochem. 226, 267–276.
11. Haga, K. & Haga, T. (1992) Activation by G protein beta gamma
subunits of agonist- or light-dependent phosphorylationof mus-
carinic acetylcholine receptors and rhodopsin. J. Biol. Chem. 267,
2222–2227.
12. Pitcher, J.A., Inglese, J., Higgins, J.B., Arriza, J.L., Casey, P.J.,
Kim, C., Benovic, J.L., Kwatra, M.M., Caron, M.G. & Lefkowitz,
R.J. (1992) Role of bc subunits of G proteins in targeting the beta-
adrenergic receptorkinase to membrane-bound receptors. Science
257, 1264–1267.
13. Kameyama, K., Haga, K., Haga, T., Kontani, K., Katada, T. &
Fukada, Y. (1993) Activation by G protein beta gamma subunits
of beta-adrenergic and muscarinic receptor kinase. J. Biol. Chem.
268, 7753–7758.
14. Haga, K., Kameyama, K. & Haga, T. (1994) Synergistic activation
of a G protein-coupled receptorkinaseby G protein beta gamma
subunits and mastoparan or related peptides. J. Biol. Chem. 269,
12594–12599.
15. Palczewski, K., Buczylko, J., Kaplan, M.W., Polans, A.S. &
Crabb, J.W. (1991) Mechanism of rhodopsin kinase activation.
J. Biol. Chem. 266, 12949–12955.
16. Moro, O., Lameh, J., Hogger, P. & Sadee, W. (1993) Hydrophobic
amino acid in the i2 loop plays a key role in receptor-G protein
coupling. J. Biol. Chem. 268, 22273–22276.
17. Wess,J.,Liu,J.,Blin,N.,Yun,J.,Lerche,C.&Kostenis,E.(1997)
Structural basis of receptor/G protein coupling selectivity studied
with muscarinic receptors as model systems. Life Sci. 60, 1007–1014.
18. Higashijima, T., Uzu, S., Nakajima, T. & Ross, E.M. (1988)
Mastoparan, a peptide toxin from wasp venom, mimics receptors
by activating GTP-binding regulatory proteins (G proteins).
J. Biol. Chem. 263, 6491–6494.
19. Haga, K., Ogawa, H., Haga, T. & Murofushi, H. (1998) GTP-
binding-protein-coupled receptorkinase2 (GRK2) binds and
phosphorylates tubulin. Eur. J. Biochem. 255, 363–368.
20. Pitcher, J.A., Hall, R.A., Daaka, Y., Zhang, J., Ferguson, S.S.,
Hester, S., Miller, S., Caron, M.G., Lefkowitz, R.J. & Barak, L.S.
(1998) The G protein-coupled receptorkinase2 is a microtubule-
associated protein kinase that phosphorylates tubulin. J. Biol.
Chem. 273, 12316–12324.
21. Carman, C.V., Som, T., Kim, C.M. & Benovic, J.L. (1998)
Binding and phosphorylationof tubulin by G protein-coupled
receptor kinases. J. Biol. Chem. 273, 20308–20316.
22. Pronin, A.N., Morris, A.J., Surguchov, A. & Benovic, J.L. (2000)
Synucleins are a novel class of substrates for G protein-coupled
receptor kinases. J. Biol. Chem. 275, 26515–26522.
23. Ruiz-Gomez, A., Humrich, J., Murga, C., Quitterer, U., Lohse,
M.J. & Mayor, F. Jr (2000) Phosphorylationof phosducin and
phosducin-like protein by G protein-coupled receptorkinase 2.
J. Biol. Chem. 275, 29724–29730.
24. Shelanski, M.L., Gaskin, F. & Cantor, C.R. (1973) Microtubule
assembly in the absence of added nucleotides. Proc. Natl Acad.
Sci. USA 70, 765–768.
25. Weingarten, M.D., Lockwood, A.H., Hwo, S.Y. & Kirschner,
M.W. (1975) A protein factor essential for microtubule assembly.
Proc.NatlAcad.Sci.USA72, 1858–1862.
26. Cresfield, A.M., Moore, S. & Stein, W.H. (1963) The preparation
and enzymatic hydrolysis of reduced and S-carboxymethylated
proteins. J. Biol. Chem. 238, 622–627.
27. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London) 227,
680–685.
28. Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl Acad. Sci.
USA 76, 4350–4354.
29. Imai, Y., Matsushima, Y., Sugimura, T. & Terada, M. (1991) A
simple and rapid method for generating a deletion by PCR.
Nucleic Acid Res. 19, 2785–2785.
30. Sullivan, K.F. (1988) Structure and utilization of tubulin isotypes.
Annu. Rev. Cell. Biol. 4, 687–716.
31. Khan, I.A. & Luduena, R.F. (1996) Phosphorylationof beta III-
tubulin. Biochemistry 35, 3704–3711.
32. Hiraga, A. & Tamura, S. (2000) Protein phosphatase 2A is asso-
ciated in an inactive state with microtubules through 2A1-specific
interaction with tubulin. Biochem. J. 346, 433–439.
33. Luduena, R.F., Zimmermann, H.P. & Little, M. (1988) Identifi-
cation of the phosphorylated beta-tubulin isotype in differentiated
neuroblastoma cells. FEBS Lett. 230, 142–146.
34. Diaz-Nido, J., Serrano, L., Lopez-Otin, C., Vandekerckhove, J. &
Avila, J. (1990) Phosphorylationof a neuronal-specific beta-
tubulin isotype. J. Biol. Chem. 265, 13949–13954.
35. Serrano, L., Diaz-Nido, J., Wandosell, F. & Avila, J. (1987)
Tubulin phosphorylationby casein kinase II is similar to that
found in vivo. J. Cell. Biol. 105, 1731–1739.
36. Pals-Rylaarsdam, R., Gurevich, V.V., Lee, K.B., Ptasienski, J.A.,
Benovic, J.L. & Hosey, M.M. (1997) Internalization of the m2
muscarinic acetylcholine receptor. Arrestin-independent and
-dependent pathways. J. Biol. Chem. 272, 23682–23689.
37. Eason, M.G., Moreira, S.P. & Liggett, S.B. (1995) Four
consecutive serines in the third intracellular loop are the sites for
b-adrenergic receptor kinase-mediated phosphorylation and
desensitization of the a
2A
-adrenergic receptor. J. Biol. Chem. 270,
4681–4688.
38. Nogales, E., Wolf, S.G. & Downing, K.H. (1998) Structure of the
alpha beta tubulin dimer by electron crystallography. Nature
(London) 391, 199–203.
39. Serrano, L., Avila, J. & Maccioni, R.B. (1984) Controlled pro-
teolysis of tubulin by subtilisin: localization of the site for MAP2
interaction. Biochemistry 23, 4675–4681.
1162 N. Yoshida et al.(Eur. J. Biochem. 270) Ó FEBS 2003
40. Wang, Z. & Sheetz, M.P. (2000) The C-terminus of tubulin
increases cytoplasmic dynein and kinesin processivity. Biophys.
J. 78, 1955–1964.
41. Wang, N., Yan, K. & Rasenick, M.M. (1990) Tubulin binds
specifically to the signal-transducing proteins, Gs alpha and Gi
alpha 1. J. Biol. Chem. 265, 1239–1242.
42. Popova, J.S., Garrison, J.C., Rhee, S.G. & Rasenick, M.M. (1997)
Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact
to regulate phospholipase Cbeta1 signaling. J. Biol. Chem. 272,
6760–6765.
43. Roychowdhury, S. & Rasenick, M.M. (1997) G protein beta1-
gamma2 subunits promote microtubule assembly. J. Biol. Chem.
272, 31576–31581.
44. Popova, J.S. & Rasenick, M.M. (2000) Muscarinic receptor acti-
vation promotes the membrane association of tubulin for the
regulation of Gq-mediated phospholipase Cbeta (1) signaling.
J. Neurosci. 20, 2774–2782.
45. Limas, C.J. & Limas, C. (1985) Carbachol induces desensitization
of cardiac beta-adrenergic receptors through muscarinic M1
receptors. Biochem. Biophys. Res. Commun. 128, 699–704.
Ó FEBS 2003 Sitesofphosphorylationby GRK2 in tubulin (Eur. J. Biochem. 270) 1163
. (1994)
Activation of a GTP-binding protein and a GTP-binding-protein-
coupled receptor kinase (b-adrenergic -receptor kinase- 1) by a
muscarinic receptor m2 mutant lacking. light-dependent phosphorylation of mus-
carinic acetylcholine receptors and rhodopsin. J. Biol. Chem. 26 7,
22 22 22 27.
12. Pitcher, J.A., Inglese, J., Higgins, J.B.,