Vol.
168,
No.
April
30,
1990
2, 1990
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages 616-624
EXPRESSION,
Roland
BIOCHEMICAL
PURIFICATION AND CRARACTERIZATION OF A 41 IcDa INSULIN RECEPTOR TYROSINR KINASE DOMAIN G. Kallen,
Joseph
E.
Smith,
Zufang
Sheng
and Lim
Tung
Protein Chemistry Group and the Department of Biochemistry Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Received
and
March 6, 1990
An active 41 kDa cytoplasmic domain of the insulin receptor tyrosine the kinase(CIRK-41) encompassing amino acid residues 946 and 1303 of native protein with an additional three amino acids(HAI) at the using N-terminus has been overexpressed the baculovirus pAC 373 expression system. The recombinant protein termed CIRK-41 has been purified to homogeneity. CIRK-41 was capable of autophosphorylation and up to 1.9 moles of phosphate were incorporated per mole of enzyme when it was incubated in the presence of 10 mM manganese chloride and resulted in stimulation of CIRK-41 0.5 mM ATP. Autophosphorylation indicating that CIRK-41 activity towards its exogenous substrate, may as a model molecule to study the role of phosphorylation and be used receptor tyrosine dephosphorylation in the control of the insulin kinase activity. 01990 Rcademic Press, Inc. In shown
response
to
undergo of
Abolition with
several
receptor
the its
serine
is
receptor
on
phosphorylation 1151
of alters
phosphotyrosyl phosphorylation
the its
protein of
these
also
present
insulin
kinase tyrosine
the
phosphorylation
to
receptor
at
and that
may [17-191.
insulin role
in
indicate
tyrosine the
on
of
results
evidence
616
the
While
of
residues
insulin protein
of
residues
activity
of
by autophosphorylation
unclear,
conformation
been
interferes
phosphotyrosyl
observed[9,12].
lines
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
its
followed
cOo6291x/90 $1.50 Copyright All rights
activity
The binding
of
tyrosine
Several
kinase
have
residues[l-41.
Phosphorylation is
at
proteins tyrosine
ce11[5-71.
then
residues
phosphorylation
and
the
by activation is
on
tyrosine
residues[9-111.
activation[l3-181.
1150
in
which
on serine
insulin
receptor
accompanied
intracellular
phosphorylation
action
tyrosine
receptor
several
insulin
activity
several
insulin,
enhanced
of
is
kinase
to
be
correlated Phosphorylation
the its that
residues
activation
of
1146, of
the with at
Vol.
168, No. 2, 1990
tyrosine
residues
correlated near
the
contribution insulin The
1316 and
with
located
receptor
in the
control
kinase
would
material.
As
overexpressed
N terminus
are
each
the
activity.
In
AND BIOPHYSICAL
located the
kinase of multisite
of
the
activity
also
reaction
of the
first
step
towards
ability this procedure
insulin to
report,
is
in
sites
The the
at the
the
control
present
insulin
availability
exact of the
unclear.
achieving
receptor
describe
and some of
its
EXPERIMENTAL
receptor of
and its
tyrosine
large
that
tyrosine
autophosphorylate we
tyrosine
be
phosphorylation/dephosphorylation
from
41KDa
Other
cannot
phosphorylated[l8,19]-
benefit a
C-terminus
enzyme[l7,20].
activity
role
RESEARCH COMMUNICATIONS
at the
phosphorylation
the
a
retains
reproducible
of
tyrosine
of
1322
activity
of
study
BIOCHEMICAL
kinase
amounts
goal,
domain
autoregulate
purification
we
of have
which its
by a highly
properties.
PROCEDURES
Materials. bovine
Fast Q Sepharose, Poly-L-Lysine-Agarose, Sephacryl S-200, serum albumin, ovalbumin, carbonic anhydrase, Glu4Tyrl Adenosine-5' -Triphosphate were from Sigma(St. Louis, MO). ~84pY;l~~~P was from England New Nuclear/Dupont(Boston, MA). Restriction enzymes for DNA work were from England New Biolabs(Beverly, MA) or Boehringer Manheim Biochemicals or Bethesda Research Laboratories(Gaithersburg, MA). EX-CELL 400 medium was from J.R. Scientific(Woodland, CA). Polyclonal antibodies directed against the b subunit of the insulin receptor were kindly provided to us by Dr. Ora M. Rosen of Memorial Sloan-Kettering Cancer Center, New York. The Spodoptera frugiperda(Sf9) cells, baculovirus AcPNV and the transfer vector, pAC373 were generously supplied by Dr. Max Summers of Texas A and M University, Texas. All other chemicals were of the highest grade Construction of recombinant baculovirus containing the gene codinq for a 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase tripartite ligation was performed with a 1.4 kb (CIRK-41). A BglI(2968)-EcoRI(pUC12 polylinker site) cDNA fragment from pUC!l2-HIR/PstI[21], two annealed oligonucleotides(oligo #l = 5'-GATCCATGCATGCGATCGAT-3'; oligo #2 = 5'-GATCGCATGCATG-3') and gel purifiedpUC8 containing BamHI and EcoRI ends. The oligonucleotides contain cohesive ends to the BamHI and BglI sites, an ATG H~;;!J2~a;~~~ initiation codon and create N-terminus a synthetic the intact human distal to the hydrophobic sequence that anchors insulin receptor to the plasma membrane. The resulting recombinant (pUC8-HIR/BamHI) encoded the cytoplasmic domain of the human insulin one at the 5'end provided by the receptor flanked by two BamHI sites: oligonucleotide linkers and one at the 3'end from the polylinker of with NciI(4164) and XbaI pUC12-HIR/PstI. pUC8-HIR/BamHI was digested (site within the polylinker portion of pUC12), rendered flush ended by treatment with Klenow DNA polymerase 1 and dNTPs, gel purified and 617
Vof. 168, No. 2, 1990
BlOCfiEMfCAL
AND BfOPHYSlCAL RESEARCH COMMUNfCATlONS
ligated. The recombinant pUC8-CIRK-41 causes insulin receptor tyrosine domain(CIRK-41) kinase BamHI fragment was inserted into the BamHI site of pAC373 located 50 base pair downstream of the polyhedrin transcription initiation or CAP site to create pAC373/CIRK-41. Transfer of the 1.4Kb fragment from pAC373/CIRK-41 to the AcNPV genome was achieved by homologous recombination into the host following calcium polyhedrin gene phosphate transfection with wild AcPNV DNA into Sf9 cells. type Recombinant viruses were plaque-purified by plaque hybridization, visual and sib screening[22]. One recombinant baculovirus termed baculovirus AcPNV/CIRK-41 containing the gene coding for insulin receptor tyrosine kinase domain encompassing 946 to 1303 residues of the human insulin receptor[21] plus a synthetic has HA1 N-terminus been purified. Culture of cytoplasmic Sf9 cells recombinant Smith[22].
Spodoptera fruqiperda(sf9) cells and expression of 41 kDa domain of the insulin receptor tyrosine kinase (CIRK-41). were grown in EX-CELL 400 medium and infected with baculovirus AcPNV/CIRK-41 as described by Summers and
Assay of 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase(CIRK-41). Assay of this enzyme was based on its ability to phosphorylate Glu4Tyrl Polymer. The reaction mixture(lOOu1) consisted of the enzyme at an appropriate dilution in 50 mM Imidazole.Cl pH 7.3, 0.2 mM EGTA, 10 mM3yanganese hloride, 0.1% (v/v) 5 to 2-mercaptoethanol and 0.1 P]-ATP(10 lo6 cpm per mM [ nanomole of ATP) and 1 mg ml Glu4Tyr Polymer. The reacion mixture was incubated at 15 and 30 6 c for min then spotted onto phosphocellulose p81 paper which was then washed extensively in 10% 'd and 1% (v/v) phosphoric acid. After drying at (w'v~oo~ric~~~~~~~~~~~,a~~P-phosphate incorporated onto Glu4Tyrl Polymer was determined by Cerenkov counting. Autophosphorylation reaction was performed as described above except that the Glu4Tyrl Polymer was omitted. Protein determination. of Bradford[23].
Protein
was determined
routinely
by the
methods
Polyacrylamide qel electrophoresis and autoradiography. SDS-PAGE was performed according to Laemmli[24]. Agarose gel electrophoresis was carried out as described in [25]. When radiolabeled proteins were used, the gels were dried and autoradiographed with x-ray film and intensifying screen. Immunoblottinq.
Immunoblotting
was performed
as described
in
[26].
Purification of a 41 kDa cytoplasmic domain of the insulin receptor tvrosine kinase(CIRK-41). Sf9 cells (Two liters) infected with recombinant baculovirus AcPNV/CIRK-41 were collected by centrifugation at 4000xg for 15 min in 6x1000 ml rotors and then sonicated in 100 ml of 50 mM Imidazole-Cl pH 7.3, 50 mM beta-glycerophosphate pH 7.3, 2mM EDTA, 2 mM EGTA, 1 mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol and 10 % (v/v) glycerol. $he homogenate was then centrifuged at 30000x g for 30 min at 2 C in 12x50 ml rotors. The supernatant was diluted two fold in 25 mM Imidazole-Cl ph 7.3, 0.2 mM EGTA, 1 mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol and 10% (v/v) glycerol (Buffer A) and loaded onto a FAST Q column (1.5 x 25 cm) equilibrated in Buffer A. The column was washed with Buffer A plus 100 mM NaCl and eluted with a 600 ml linear gradient of Buffer A plus 100 mM NaCl to Buffer A plus 300 mM NaCl. The active fractions eluting at about 250 mM NaCl salt concentration were collected, 618
Vol.
168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
diluted two fold with Buffer A and loaded onto a Poly-L-Lysine Agarose column equilibrated in Buffer A. The column was washed with Buffer A plus 50 mM NaCl and developed with a 400 mL linear gradient of Buffer A plus 50 mM NaCl to Buffer A plus 400 mM NaCl. The active fractions eluting at about 250 mM NaCl salt concentration were pooled, concentrated to about 5 ml by vacuum dialysis and loaded onto a Sephacryl S-200 column equilibrated in 50 mM Imidazole-Cl pH 7.3, 0.2 mM EGTA, 1mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol, 0.2 M NaCl and 10 % (v/v) glycerol. The active fraction eluting with an apparent molecular mass of 45KDa was pooled, concentrated and stored at -2O'C in the presence of 60 % (v/v) glycerol. RESULTS AND DISCUSSION The kinase
41
kDa
termed
cytosolic
CIRK-41
fraction
AcPNV/CIRK-41. and
on Fast
to gel
enzyme
molecular
Sf9 cells
its
against
on calibrated
with
the
(Figure
1, panel
l.Extract 2.Fast Q 3.POlY -L-Lysine 4.Sephacryl s-200
B). and
ions
described
(ml 1
by
Protein
[
it
was shown).
apparent
(Figure
using
an antibody that
insulin
was
41 kDa protein
cytoplasmic domain of the insulin 2 liters of Sf9 cells infected was used. Activity was assayed as in materials and methods
(mg) (units/ml)
Specific activity (units/mg)
Purification
Yield (%I
130 39
1015.0 94.0
4058 2405
3.9 25.6
1.0 6.6
100 59
37
21.9
804
36.7
9.4
20
21
a.7
515
59.2
15.1
13
619
41
receptor
preparation the
1,
the
kDa
Activity
as
PAGE
confirmed
32P]-ATP,
1
eluted
(not
band of
SDS
enzyme
Table
when
S-200.
protein
the
chromatography
45KDa
domain of the When the
in
CIRK-41
analysis
receptor[27]
cytoplasmic
manganese
Volume
major
immunoblotting
Table 1. Purification of 41 receptor tyrosine kinase(CIRK-41). with baculovirus AcPNV/CIRK-41 Steps
a
the human insulin
S-200,
in
baculovirus
summarized
Sephacryl
was analysed
Western
recombinant
mass
tyrosine
protein
by successive
molecular
exhibited
was indeed
kinase
incubated
4).
is
and Sephacryl
apparent
receptor
soluble
with
purification
filtration
insulin
a
infected
Agarose
of
as
of CIRK-41
mass 41 kDa when it
protein
tyrosine
recovered
preparation
A, lane
directed
domain of the
The purification
protein
subjected
kDa
of
Q, Poly-L-lysine
a globular
panel
was
1. Following
Figure
The
cytoplasmic
Vol.
168,
No.
BIOCHEMICAL
2, 1990
A
AND
BIOPHYSICAL
RESEARCH
B
COMMUNICATIONS
C
29.5-
29.5-,
Fiq. 1. Purification of the 41KDa cytoplasmic domain of the insulin receptor tyrosine kinase(CIRK-41). Panel A, SDS PAGE pattern of pooled fractions at each step of purification. The gel was stained with Coomassie blue. Lanes M, serum protein standards, from top to bottom, phosphorylase b, bovine carbonic anhydrase and soy bean trypsin inhibitor; albumin, ovalbumin, Lane 2, Past Q; Lane 3, Poly-L-Lysine Agarose; Lane Lane 1, Extract: 4, Sephacryl S-200. Panel S, Immunoblot analysis of pooled fractions at each step of purification. Lanes 1 to 4 correspond to the lanes in Panel A. Panel C, Autophosphorylation of CIRK-41 in pooled fractions at each step of purification. Lanes 1 to 4 correspond to the lanes in Panel A.
underwent
autophosphorylation.
molecular
masses
Apparently
phosphorylation
PAGE. (Figure resistant
slightly
treatment
phosphorylated
of
on with
the
CIRK-41 4).
of
55'~
molecular CIRK-41
Like
native
autophosphorylated phosphate
at
was
and
autophosphorylation manganese ions,
receptor
relatively
was 0.5
mM ATP was lower
magnesium ions
above
mass
of
results
2).
was was
are
all
of
CIRK-41
and up to of
Although
25
1).
kinase,
presence
was nevertheless
it
SDS
10 mM manganese
(Table
when magnesium ions
620
that
activity
stoichiometry mol
on
CIRK-41.
reaction
in the
(Figure
observed.
CIRK-41
of substrate,
per
achieved
also
confirming
tyrosine
high
incorporated
autophosphorylation chloride
insulin
apparent
mobility
had a specific
and 0.1 mM ATP were used in the the
were
its
The
per mg of enzyme when 1 mg per ml
chloride
kDa
of
Phosphorylated
residues.
theoretical
41
altered
1M KOH at
tyrosine
species
than
C, lane
with
Homogenous preparation units
higher
1, panel
to
consistent
Phosphorylated
enzyme. of
was
1.9 mol Maximal
10 mM manganese the
extent
was used instead
an effective
cofactor
of of
Vol. 166, No. 2, 1990
for
the
BIOCHEMICAL
autophosphorylation receptor
tyrosine
CIRK-41
resulted
in activation
initial
Maximal
rate
two
fold.
after
of There
tyrosine
ligand
kinase
tyrosine
is
the
implicated
its
(figure binding,
tyrosine
kinase
these
sites
the
towards
resulted
in an increase
its
its
of
activity
of
native
Vmax value change
exogenous of
by
in its
its
nearly Km value
4). the
regulated
sites
Like
autophosphorylation
no significant
activity
of
the
insulin
by autophosphorylation
residues[11,13,14,18,19].
phosphorylation
within
of
was apparently
3).
domain,
and an increase
autophosphorylation Following
Of
kinase
autophosphorylation
reaction
RESEARCH COMMUNICATIONS
reaction(figure
insulin
substrates.
AND BIOPHYSICAL
Of particular located
at
catalytic as the
on several
interest
positions domain.
phosphorylation
receptor
is
1146,
a
key
series
1150 and 1151
Several
studies
have
sites
responsible
for
2.0 is! ET 5 1.5 5 E 3
1.0
L 8 g
0.5
5
E 0.0
02
5
Time (min)
10
03
a004
1
0
I
.
5
I
10
divalent ions (mM)
Fig. 2. Time course of the autophosphorylation of CIRK-41. The enzyme(0.004 mq) from the final ourification steu was incubated at 3006 in 50 -mM Imidazole-Cl -pH 7.3, 0.2 mM- EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 % (v/v) glycerol, 10 m manganese chloride and 0.1 mM (squares), or 0.5 mM (circles) ['YY2PI-ATP. At the indicated times, the reaction mixture was heated at 100°C for 3 min with an equal volume of denaturing sample buffer and subjected to SDS PAGE. T&e gels were then stained with Coomassie blue, destained and dried. onto CIRK-41 was determined by excising the band P incorporation corresponding to CIRK-41 and counting the gel slice by Cerenkov method. cations Fig. 3. Effects of divalent on the autophosphorylation of CIRK-41. CIRK-41 was incubated for 10 min in the presence of 0.1 mMp";iyfigq~]-ATP and different concentrations of manganese chloride(circles) or magnesium chloride(squares). 621
BIOCHEMICAL
Vol. 168, No. 2, 1990
AND BIOPHYSICAL
time (min)
RESEARCH COMMUNICATIONS
Substrate concentration (mg~ml)
of autophosphorylation on the activity of CIRK towards the last substrates. 0.03 mg of CIRK-41 from purification step was preincubated for 5 min in 50 mM Imidazole-Cl pH 7.3, 50 mM beta-glycerophosphate pH 7.3, 0.2 mM EGTA, 0.1 % (v/v) 10 mM manganese chloride and 2-mercaptoethanol, 10 % (v/v) glycerol, incubation was performed in which 0.5 mM unlabeled ATP. A control The incubation mixtures were then diluted unlabeled ATP was ommitted. ATP. 0.01 lo-fold in the above buffer without manganese chloride and ml of the diluted enzyme ~3: then assayed in the presence of 10 mM ]-ATP and substrate as described in manganese chloride, 0.1 mM [Y materials and methods. the phosphorylation of exogenous Panel A, Time course of substrate by phospho(circles) and dephospho(squares) CIRK-41. of different concentrations of exogenous Panel B, Phosphorylation substrates by phospho(circles) and dephospho(squares) CIRK-41.
p$A-
Effects
exogenous
controlling role
of
although
the
activity
phosphorylation
C terminus
of
the
on tyrosine
kinase of
residues
953 and 960.
sites
obscure[27,28].
To
simplify
the
can also
the
study
of
the
tyrosine,
to
express
receptor for
sought
tyrosine control
of
phosphorylation truncated
kinase its
sites.
versions
of
a
activity
the
role
the
to affect
the
Substantial ATP into
tyrosine
of multisite
phosphorylation
of
minimum
insulin
the size,
devoid
receptor
in
receptor
soluble
phosphorylation
been successful
622
appear
at
removal
phosphorylation
and
insulin
Thus
latter
regulation
domain with
We have
of
the
in
proteolytic
from
The
occurs
enzyme[17,20,27,28].
role
and dephosphorylation we
does not
be incorporated
the
kinase.
incorporation
1316 and 1322,
residues
of
tyrosine
have been more ambiguous.
of phosphate
those
Again
receptor
sites
residues
activity
phosphate
insulin
other
portion
domain containing
tyrosine
is
at
a substantial
the
amount
of the
sites of
non
insulin necessary essential
expressing
tyrosine
kinase
several domain
Vol.
168, No. 2, 1990
lacking
the
the
terminus.
C
non essential
cytoplasmic
in this
which
Truncation tyrosine
kinase
did
autophosphorylate substrate.
did
and
This
role
control
of
the
interfere
is
insulin
with
its
in
CIRK-41
of multisite
kinase
is
an ideal
phosphorylation receptor
kinase,
sites
1316
of
the
insulin
the
ability
CIRK-41,
946 to
1303
lacks
the
and therefore
and
of CIRK-41
other
model protein
kinase
to
an exogenous documented for
the
and dephosphorylation
tyrosine
1322.
receptor
towards with
at
recombinant
residues
activity
agreement
located
active
tyrosine
region
regulate
result
studies[17,20,27,28]. of the
not
smallest
phosphorylation
particular
sites
encompasses
tyrosine
the
that
the
receptor
CIRK-41
receptor
contains
of
of
insulin
report.
RESEARCH COMMUNICATIONS
phosphorylation
The purification
the human insulin
C terminus
AND BIOPHYSICAL
tyrosine
domain of the
is described of
BIOCHEMICAL
study
in
the
activity.
ACKNOWLEDGMENTS This University Center
of
Michael
from
was supported
Pennsylvania
which
Pennsylvania Grants
work
is
the
NIH.
Wong in the
School
funded
Research
by a Pilot
by
NIH
Medicine's
Grant
Foundation
We appreciate initial
of
and Feasibility
and the
phase of
DK 19525, Biomedical
participation
this
Grant
Diabetes
of
Research
the University Research of
the
of
Support
Adam Cohen and
project.
REFERENCES 1. White, M. F., Maron, C. R. and Kahn, R. (1985) Nature. 318, 1832. Rees-Jones, R. W. and Taylor, S. I. (1985) J. Biol. Chem. 246, 4461-4467. 3. Bernier, M., Laird, D. M. and Lane M. D. (1988) J. Biol. Chem. 263, 13626-13634. 4. KaraSik, A., Pepinsky, R. B., Shoelson, S. E. and Kahn, R. C. (1988) J. Biol. Chem. 262, 11862-11867. 5. Morgan, D. O., Ho. L., Korn, L. J. and Roth, R. A. (1986) Proc. Natl. Acad. Sci. (USA). 83, 328-332. 6. Russel, D. S., Gherzi, R., Johnson, E. L., Chop, C. K. and Rosen,O. M. (1987) J. Biol. Chem. 262, 11833-11840. 7. Chou, C. K., Dull, T. J., RUssel, D. S., Gherzi, R., Lebwolh, D. Ullrich, A. and Rosen, 0. M. (1987) J. Biol. Chem. 262, 1842-1847. 8. IZUmi, T., Sakai, Y., Akanuma, Y., Takaku, F. and KGga, M. (1988) J. Biol. Chem. 263, 10386-10393. 9. Kasuga, M., Karlsson, T. A. and Kahn, C. R. (1982) Science. 2, 185-187. 623
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RESEARCH COMMUNICATIONS
Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D. L. and Kahn, C. R. (1983) Proc. Natl. Acad. Sci. (USA) 80, 2137-2141. 11. Rosen, 0. M. (1987) Science. 237, 1452-1458. 12. Smith, D. M., King, M. J. andxlade, G. J. (1988) Biochem. J. 250 509-519. 13. Petruzelli, L., Herrera, R. and Rosen, 0. M. (1984) Proc. Natl. Acad. (USA) 81, 3327-3331. 14. Rosen, 0. M., Herrera, R., Olowe, Y., Petruzelli, L. M. and Cobb, M. H. (1983) Proc. Natl. Acad. Sci. (USA) 80, 3237-3240. 15. Yu, K. T. and Czech, M. P. (1984) J. Biol. Chem. 259, 5277-5286. 16. Kwok, Y. C., Nemenoff, R. A., Powers, A. C. and Avruch, J. (1986) Arch. Biochem. Biophys. 244, 102-113. Herrera, R. and Rosen, 0. M. (1986) J. Biol. Chem. 261, 17. 11980-11985. 18. Tornquist, H. E., Pierce, M. W., Frackelton, A. R., Nemenoff, R. A. and Avruch, J. (1987) J. Biol. Chem. 262, 10212-10219. 19. White, M. F., Shoelson, S. E., Kentmann, H. and Kahn, C. R. (1988) J. Biol. Chem. 263, 2969-2980. Goren, H. J., m-e, M. F. and Kahn, C. R. (1987) Biochemistry. 20. 10.
26, 21.
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23. 24. 25.
26. 27.
28. 29. 30.
2374-2382.
Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzelli, L. Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfield, D., Rosen, 0. M. and Ramachandran, J. (1985) Nature. 313, 756-767. Summers, M. D. and Smith, G. E. (1987) Manual of Methods for Baculovirus and Insect Cell Culture Procedures. (Texas A & M Univ. Press, College station, TX) Bradford, M. M. (1976) Anal Biochem. 72, 248-254. Laemmli, U. K. (1970) Nature. 227, 680-685. Maniatis, T., Fritsh, E. F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Tung, L. and Reed, L. J. (1989) J. Biol. Chem. 264, 2985-2990. Herrera, R., Petruzelli, L. M., Thomas, N., BraGn, H. N., Kaiser, E. T. and Rosen, 0. M. (1985) Proc. Natl. Acad. Sci. (USA) 82, 7899-7903. Herrera, R. Lebwohl, D. Garcia de Herreros, A., Kallen, R. G. and Rosen, 0. M. (1988) J. Biol. Chem. 263, 5560-5568. Tornquist, H. E. and Avruch, J. (1988) J. Biol. Chem. 263, 4593-4601. White, M. F., Livingston, J. N., Becker, J. M., Lavris, V., Dull, A. and Kahn, C. R. (1988) Cell. 54, 641-649. T. J., Ullrich,
624
168,
No.
April
30,
1990
2, 1990
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages 616-624
EXPRESSION,
Roland
BIOCHEMICAL
PURIFICATION AND CRARACTERIZATION OF A 41 IcDa INSULIN RECEPTOR TYROSINR KINASE DOMAIN G. Kallen,
Joseph
E.
Smith,
Zufang
Sheng
and Lim
Tung
Protein Chemistry Group and the Department of Biochemistry Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Received
and
March 6, 1990
An active 41 kDa cytoplasmic domain of the insulin receptor tyrosine the kinase(CIRK-41) encompassing amino acid residues 946 and 1303 of native protein with an additional three amino acids(HAI) at the using N-terminus has been overexpressed the baculovirus pAC 373 expression system. The recombinant protein termed CIRK-41 has been purified to homogeneity. CIRK-41 was capable of autophosphorylation and up to 1.9 moles of phosphate were incorporated per mole of enzyme when it was incubated in the presence of 10 mM manganese chloride and resulted in stimulation of CIRK-41 0.5 mM ATP. Autophosphorylation indicating that CIRK-41 activity towards its exogenous substrate, may as a model molecule to study the role of phosphorylation and be used receptor tyrosine dephosphorylation in the control of the insulin kinase activity. 01990 Rcademic Press, Inc. In shown
response
to
undergo of
Abolition with
several
receptor
the its
serine
is
receptor
on
phosphorylation 1151
of alters
phosphotyrosyl phosphorylation
the its
protein of
these
also
present
insulin
kinase tyrosine
the
phosphorylation
to
receptor
at
and that
may [17-191.
insulin role
in
indicate
tyrosine the
on
of
results
evidence
616
the
While
of
residues
insulin protein
of
residues
activity
of
by autophosphorylation
unclear,
conformation
been
interferes
phosphotyrosyl
observed[9,12].
lines
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
its
followed
cOo6291x/90 $1.50 Copyright All rights
activity
The binding
of
tyrosine
Several
kinase
have
residues[l-41.
Phosphorylation is
at
proteins tyrosine
ce11[5-71.
then
residues
phosphorylation
and
the
by activation is
on
tyrosine
residues[9-111.
activation[l3-181.
1150
in
which
on serine
insulin
receptor
accompanied
intracellular
phosphorylation
action
tyrosine
receptor
several
insulin
activity
several
insulin,
enhanced
of
is
kinase
to
be
correlated Phosphorylation
the its that
residues
activation
of
1146, of
the with at
Vol.
168, No. 2, 1990
tyrosine
residues
correlated near
the
contribution insulin The
1316 and
with
located
receptor
in the
control
kinase
would
material.
As
overexpressed
N terminus
are
each
the
activity.
In
AND BIOPHYSICAL
located the
kinase of multisite
of
the
activity
also
reaction
of the
first
step
towards
ability this procedure
insulin to
report,
is
in
sites
The the
at the
the
control
present
insulin
availability
exact of the
unclear.
achieving
receptor
describe
and some of
its
EXPERIMENTAL
receptor of
and its
tyrosine
large
that
tyrosine
autophosphorylate we
tyrosine
be
phosphorylation/dephosphorylation
from
41KDa
Other
cannot
phosphorylated[l8,19]-
benefit a
C-terminus
enzyme[l7,20].
activity
role
RESEARCH COMMUNICATIONS
at the
phosphorylation
the
a
retains
reproducible
of
tyrosine
of
1322
activity
of
study
BIOCHEMICAL
kinase
amounts
goal,
domain
autoregulate
purification
we
of have
which its
by a highly
properties.
PROCEDURES
Materials. bovine
Fast Q Sepharose, Poly-L-Lysine-Agarose, Sephacryl S-200, serum albumin, ovalbumin, carbonic anhydrase, Glu4Tyrl Adenosine-5' -Triphosphate were from Sigma(St. Louis, MO). ~84pY;l~~~P was from England New Nuclear/Dupont(Boston, MA). Restriction enzymes for DNA work were from England New Biolabs(Beverly, MA) or Boehringer Manheim Biochemicals or Bethesda Research Laboratories(Gaithersburg, MA). EX-CELL 400 medium was from J.R. Scientific(Woodland, CA). Polyclonal antibodies directed against the b subunit of the insulin receptor were kindly provided to us by Dr. Ora M. Rosen of Memorial Sloan-Kettering Cancer Center, New York. The Spodoptera frugiperda(Sf9) cells, baculovirus AcPNV and the transfer vector, pAC373 were generously supplied by Dr. Max Summers of Texas A and M University, Texas. All other chemicals were of the highest grade Construction of recombinant baculovirus containing the gene codinq for a 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase tripartite ligation was performed with a 1.4 kb (CIRK-41). A BglI(2968)-EcoRI(pUC12 polylinker site) cDNA fragment from pUC!l2-HIR/PstI[21], two annealed oligonucleotides(oligo #l = 5'-GATCCATGCATGCGATCGAT-3'; oligo #2 = 5'-GATCGCATGCATG-3') and gel purifiedpUC8 containing BamHI and EcoRI ends. The oligonucleotides contain cohesive ends to the BamHI and BglI sites, an ATG H~;;!J2~a;~~~ initiation codon and create N-terminus a synthetic the intact human distal to the hydrophobic sequence that anchors insulin receptor to the plasma membrane. The resulting recombinant (pUC8-HIR/BamHI) encoded the cytoplasmic domain of the human insulin one at the 5'end provided by the receptor flanked by two BamHI sites: oligonucleotide linkers and one at the 3'end from the polylinker of with NciI(4164) and XbaI pUC12-HIR/PstI. pUC8-HIR/BamHI was digested (site within the polylinker portion of pUC12), rendered flush ended by treatment with Klenow DNA polymerase 1 and dNTPs, gel purified and 617
Vof. 168, No. 2, 1990
BlOCfiEMfCAL
AND BfOPHYSlCAL RESEARCH COMMUNfCATlONS
ligated. The recombinant pUC8-CIRK-41 causes insulin receptor tyrosine domain(CIRK-41) kinase BamHI fragment was inserted into the BamHI site of pAC373 located 50 base pair downstream of the polyhedrin transcription initiation or CAP site to create pAC373/CIRK-41. Transfer of the 1.4Kb fragment from pAC373/CIRK-41 to the AcNPV genome was achieved by homologous recombination into the host following calcium polyhedrin gene phosphate transfection with wild AcPNV DNA into Sf9 cells. type Recombinant viruses were plaque-purified by plaque hybridization, visual and sib screening[22]. One recombinant baculovirus termed baculovirus AcPNV/CIRK-41 containing the gene coding for insulin receptor tyrosine kinase domain encompassing 946 to 1303 residues of the human insulin receptor[21] plus a synthetic has HA1 N-terminus been purified. Culture of cytoplasmic Sf9 cells recombinant Smith[22].
Spodoptera fruqiperda(sf9) cells and expression of 41 kDa domain of the insulin receptor tyrosine kinase (CIRK-41). were grown in EX-CELL 400 medium and infected with baculovirus AcPNV/CIRK-41 as described by Summers and
Assay of 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase(CIRK-41). Assay of this enzyme was based on its ability to phosphorylate Glu4Tyrl Polymer. The reaction mixture(lOOu1) consisted of the enzyme at an appropriate dilution in 50 mM Imidazole.Cl pH 7.3, 0.2 mM EGTA, 10 mM3yanganese hloride, 0.1% (v/v) 5 to 2-mercaptoethanol and 0.1 P]-ATP(10 lo6 cpm per mM [ nanomole of ATP) and 1 mg ml Glu4Tyr Polymer. The reacion mixture was incubated at 15 and 30 6 c for min then spotted onto phosphocellulose p81 paper which was then washed extensively in 10% 'd and 1% (v/v) phosphoric acid. After drying at (w'v~oo~ric~~~~~~~~~~~,a~~P-phosphate incorporated onto Glu4Tyrl Polymer was determined by Cerenkov counting. Autophosphorylation reaction was performed as described above except that the Glu4Tyrl Polymer was omitted. Protein determination. of Bradford[23].
Protein
was determined
routinely
by the
methods
Polyacrylamide qel electrophoresis and autoradiography. SDS-PAGE was performed according to Laemmli[24]. Agarose gel electrophoresis was carried out as described in [25]. When radiolabeled proteins were used, the gels were dried and autoradiographed with x-ray film and intensifying screen. Immunoblottinq.
Immunoblotting
was performed
as described
in
[26].
Purification of a 41 kDa cytoplasmic domain of the insulin receptor tvrosine kinase(CIRK-41). Sf9 cells (Two liters) infected with recombinant baculovirus AcPNV/CIRK-41 were collected by centrifugation at 4000xg for 15 min in 6x1000 ml rotors and then sonicated in 100 ml of 50 mM Imidazole-Cl pH 7.3, 50 mM beta-glycerophosphate pH 7.3, 2mM EDTA, 2 mM EGTA, 1 mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol and 10 % (v/v) glycerol. $he homogenate was then centrifuged at 30000x g for 30 min at 2 C in 12x50 ml rotors. The supernatant was diluted two fold in 25 mM Imidazole-Cl ph 7.3, 0.2 mM EGTA, 1 mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol and 10% (v/v) glycerol (Buffer A) and loaded onto a FAST Q column (1.5 x 25 cm) equilibrated in Buffer A. The column was washed with Buffer A plus 100 mM NaCl and eluted with a 600 ml linear gradient of Buffer A plus 100 mM NaCl to Buffer A plus 300 mM NaCl. The active fractions eluting at about 250 mM NaCl salt concentration were collected, 618
Vol.
168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
diluted two fold with Buffer A and loaded onto a Poly-L-Lysine Agarose column equilibrated in Buffer A. The column was washed with Buffer A plus 50 mM NaCl and developed with a 400 mL linear gradient of Buffer A plus 50 mM NaCl to Buffer A plus 400 mM NaCl. The active fractions eluting at about 250 mM NaCl salt concentration were pooled, concentrated to about 5 ml by vacuum dialysis and loaded onto a Sephacryl S-200 column equilibrated in 50 mM Imidazole-Cl pH 7.3, 0.2 mM EGTA, 1mM benzamidine, 0.1 mM PMSF, 0.1 % (v/v) 2-mercaptoethanol, 0.2 M NaCl and 10 % (v/v) glycerol. The active fraction eluting with an apparent molecular mass of 45KDa was pooled, concentrated and stored at -2O'C in the presence of 60 % (v/v) glycerol. RESULTS AND DISCUSSION The kinase
41
kDa
termed
cytosolic
CIRK-41
fraction
AcPNV/CIRK-41. and
on Fast
to gel
enzyme
molecular
Sf9 cells
its
against
on calibrated
with
the
(Figure
1, panel
l.Extract 2.Fast Q 3.POlY -L-Lysine 4.Sephacryl s-200
B). and
ions
described
(ml 1
by
Protein
[
it
was shown).
apparent
(Figure
using
an antibody that
insulin
was
41 kDa protein
cytoplasmic domain of the insulin 2 liters of Sf9 cells infected was used. Activity was assayed as in materials and methods
(mg) (units/ml)
Specific activity (units/mg)
Purification
Yield (%I
130 39
1015.0 94.0
4058 2405
3.9 25.6
1.0 6.6
100 59
37
21.9
804
36.7
9.4
20
21
a.7
515
59.2
15.1
13
619
41
receptor
preparation the
1,
the
kDa
Activity
as
PAGE
confirmed
32P]-ATP,
1
eluted
(not
band of
SDS
enzyme
Table
when
S-200.
protein
the
chromatography
45KDa
domain of the When the
in
CIRK-41
analysis
receptor[27]
cytoplasmic
manganese
Volume
major
immunoblotting
Table 1. Purification of 41 receptor tyrosine kinase(CIRK-41). with baculovirus AcPNV/CIRK-41 Steps
a
the human insulin
S-200,
in
baculovirus
summarized
Sephacryl
was analysed
Western
recombinant
mass
tyrosine
protein
by successive
molecular
exhibited
was indeed
kinase
incubated
4).
is
and Sephacryl
apparent
receptor
soluble
with
purification
filtration
insulin
a
infected
Agarose
of
as
of CIRK-41
mass 41 kDa when it
protein
tyrosine
recovered
preparation
A, lane
directed
domain of the
The purification
protein
subjected
kDa
of
Q, Poly-L-lysine
a globular
panel
was
1. Following
Figure
The
cytoplasmic
Vol.
168,
No.
BIOCHEMICAL
2, 1990
A
AND
BIOPHYSICAL
RESEARCH
B
COMMUNICATIONS
C
29.5-
29.5-,
Fiq. 1. Purification of the 41KDa cytoplasmic domain of the insulin receptor tyrosine kinase(CIRK-41). Panel A, SDS PAGE pattern of pooled fractions at each step of purification. The gel was stained with Coomassie blue. Lanes M, serum protein standards, from top to bottom, phosphorylase b, bovine carbonic anhydrase and soy bean trypsin inhibitor; albumin, ovalbumin, Lane 2, Past Q; Lane 3, Poly-L-Lysine Agarose; Lane Lane 1, Extract: 4, Sephacryl S-200. Panel S, Immunoblot analysis of pooled fractions at each step of purification. Lanes 1 to 4 correspond to the lanes in Panel A. Panel C, Autophosphorylation of CIRK-41 in pooled fractions at each step of purification. Lanes 1 to 4 correspond to the lanes in Panel A.
underwent
autophosphorylation.
molecular
masses
Apparently
phosphorylation
PAGE. (Figure resistant
slightly
treatment
phosphorylated
of
on with
the
CIRK-41 4).
of
55'~
molecular CIRK-41
Like
native
autophosphorylated phosphate
at
was
and
autophosphorylation manganese ions,
receptor
relatively
was 0.5
mM ATP was lower
magnesium ions
above
mass
of
results
2).
was was
are
all
of
CIRK-41
and up to of
Although
25
1).
kinase,
presence
was nevertheless
it
SDS
10 mM manganese
(Table
when magnesium ions
620
that
activity
stoichiometry mol
on
CIRK-41.
reaction
in the
(Figure
observed.
CIRK-41
of substrate,
per
achieved
also
confirming
tyrosine
high
incorporated
autophosphorylation chloride
insulin
apparent
mobility
had a specific
and 0.1 mM ATP were used in the the
were
its
The
per mg of enzyme when 1 mg per ml
chloride
kDa
of
Phosphorylated
residues.
theoretical
41
altered
1M KOH at
tyrosine
species
than
C, lane
with
Homogenous preparation units
higher
1, panel
to
consistent
Phosphorylated
enzyme. of
was
1.9 mol Maximal
10 mM manganese the
extent
was used instead
an effective
cofactor
of of
Vol. 166, No. 2, 1990
for
the
BIOCHEMICAL
autophosphorylation receptor
tyrosine
CIRK-41
resulted
in activation
initial
Maximal
rate
two
fold.
after
of There
tyrosine
ligand
kinase
tyrosine
is
the
implicated
its
(figure binding,
tyrosine
kinase
these
sites
the
towards
resulted
in an increase
its
its
of
activity
of
native
Vmax value change
exogenous of
by
in its
its
nearly Km value
4). the
regulated
sites
Like
autophosphorylation
no significant
activity
of
the
insulin
by autophosphorylation
residues[11,13,14,18,19].
phosphorylation
within
of
was apparently
3).
domain,
and an increase
autophosphorylation Following
Of
kinase
autophosphorylation
reaction
RESEARCH COMMUNICATIONS
reaction(figure
insulin
substrates.
AND BIOPHYSICAL
Of particular located
at
catalytic as the
on several
interest
positions domain.
phosphorylation
receptor
is
1146,
a
key
series
1150 and 1151
Several
studies
have
sites
responsible
for
2.0 is! ET 5 1.5 5 E 3
1.0
L 8 g
0.5
5
E 0.0
02
5
Time (min)
10
03
a004
1
0
I
.
5
I
10
divalent ions (mM)
Fig. 2. Time course of the autophosphorylation of CIRK-41. The enzyme(0.004 mq) from the final ourification steu was incubated at 3006 in 50 -mM Imidazole-Cl -pH 7.3, 0.2 mM- EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 % (v/v) glycerol, 10 m manganese chloride and 0.1 mM (squares), or 0.5 mM (circles) ['YY2PI-ATP. At the indicated times, the reaction mixture was heated at 100°C for 3 min with an equal volume of denaturing sample buffer and subjected to SDS PAGE. T&e gels were then stained with Coomassie blue, destained and dried. onto CIRK-41 was determined by excising the band P incorporation corresponding to CIRK-41 and counting the gel slice by Cerenkov method. cations Fig. 3. Effects of divalent on the autophosphorylation of CIRK-41. CIRK-41 was incubated for 10 min in the presence of 0.1 mMp";iyfigq~]-ATP and different concentrations of manganese chloride(circles) or magnesium chloride(squares). 621
BIOCHEMICAL
Vol. 168, No. 2, 1990
AND BIOPHYSICAL
time (min)
RESEARCH COMMUNICATIONS
Substrate concentration (mg~ml)
of autophosphorylation on the activity of CIRK towards the last substrates. 0.03 mg of CIRK-41 from purification step was preincubated for 5 min in 50 mM Imidazole-Cl pH 7.3, 50 mM beta-glycerophosphate pH 7.3, 0.2 mM EGTA, 0.1 % (v/v) 10 mM manganese chloride and 2-mercaptoethanol, 10 % (v/v) glycerol, incubation was performed in which 0.5 mM unlabeled ATP. A control The incubation mixtures were then diluted unlabeled ATP was ommitted. ATP. 0.01 lo-fold in the above buffer without manganese chloride and ml of the diluted enzyme ~3: then assayed in the presence of 10 mM ]-ATP and substrate as described in manganese chloride, 0.1 mM [Y materials and methods. the phosphorylation of exogenous Panel A, Time course of substrate by phospho(circles) and dephospho(squares) CIRK-41. of different concentrations of exogenous Panel B, Phosphorylation substrates by phospho(circles) and dephospho(squares) CIRK-41.
p$A-
Effects
exogenous
controlling role
of
although
the
activity
phosphorylation
C terminus
of
the
on tyrosine
kinase of
residues
953 and 960.
sites
obscure[27,28].
To
simplify
the
can also
the
study
of
the
tyrosine,
to
express
receptor for
sought
tyrosine control
of
phosphorylation truncated
kinase its
sites.
versions
of
a
activity
the
role
the
to affect
the
Substantial ATP into
tyrosine
of multisite
phosphorylation
of
minimum
insulin
the size,
devoid
receptor
in
receptor
soluble
phosphorylation
been successful
622
appear
at
removal
phosphorylation
and
insulin
Thus
latter
regulation
domain with
We have
of
the
in
proteolytic
from
The
occurs
enzyme[17,20,27,28].
role
and dephosphorylation we
does not
be incorporated
the
kinase.
incorporation
1316 and 1322,
residues
of
tyrosine
have been more ambiguous.
of phosphate
those
Again
receptor
sites
residues
activity
phosphate
insulin
other
portion
domain containing
tyrosine
is
at
a substantial
the
amount
of the
sites of
non
insulin necessary essential
expressing
tyrosine
kinase
several domain
Vol.
168, No. 2, 1990
lacking
the
the
terminus.
C
non essential
cytoplasmic
in this
which
Truncation tyrosine
kinase
did
autophosphorylate substrate.
did
and
This
role
control
of
the
interfere
is
insulin
with
its
in
CIRK-41
of multisite
kinase
is
an ideal
phosphorylation receptor
kinase,
sites
1316
of
the
insulin
the
ability
CIRK-41,
946 to
1303
lacks
the
and therefore
and
of CIRK-41
other
model protein
kinase
to
an exogenous documented for
the
and dephosphorylation
tyrosine
1322.
receptor
towards with
at
recombinant
residues
activity
agreement
located
active
tyrosine
region
regulate
result
studies[17,20,27,28]. of the
not
smallest
phosphorylation
particular
sites
encompasses
tyrosine
the
that
the
receptor
CIRK-41
receptor
contains
of
of
insulin
report.
RESEARCH COMMUNICATIONS
phosphorylation
The purification
the human insulin
C terminus
AND BIOPHYSICAL
tyrosine
domain of the
is described of
BIOCHEMICAL
study
in
the
activity.
ACKNOWLEDGMENTS This University Center
of
Michael
from
was supported
Pennsylvania
which
Pennsylvania Grants
work
is
the
NIH.
Wong in the
School
funded
Research
by a Pilot
by
NIH
Medicine's
Grant
Foundation
We appreciate initial
of
and Feasibility
and the
phase of
DK 19525, Biomedical
participation
this
Grant
Diabetes
of
Research
the University Research of
the
of
Support
Adam Cohen and
project.
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Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzelli, L. Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfield, D., Rosen, 0. M. and Ramachandran, J. (1985) Nature. 313, 756-767. Summers, M. D. and Smith, G. E. (1987) Manual of Methods for Baculovirus and Insect Cell Culture Procedures. (Texas A & M Univ. Press, College station, TX) Bradford, M. M. (1976) Anal Biochem. 72, 248-254. Laemmli, U. K. (1970) Nature. 227, 680-685. Maniatis, T., Fritsh, E. F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Tung, L. and Reed, L. J. (1989) J. Biol. Chem. 264, 2985-2990. Herrera, R., Petruzelli, L. M., Thomas, N., BraGn, H. N., Kaiser, E. T. and Rosen, 0. M. (1985) Proc. Natl. Acad. Sci. (USA) 82, 7899-7903. Herrera, R. Lebwohl, D. Garcia de Herreros, A., Kallen, R. G. and Rosen, 0. M. (1988) J. Biol. Chem. 263, 5560-5568. Tornquist, H. E. and Avruch, J. (1988) J. Biol. Chem. 263, 4593-4601. White, M. F., Livingston, J. N., Becker, J. M., Lavris, V., Dull, A. and Kahn, C. R. (1988) Cell. 54, 641-649. T. J., Ullrich,
624
