Purification and structural study of the b form of humancAMP-dependent protein kinase inhibitor Rong Jin1, Linsen Dai1, Jinbiao Zheng1and Chaoneng Ji2 1 Center of Analysis and Measuremen
Trang 1Purification and structural study of the b form of human
cAMP-dependent protein kinase inhibitor
Rong Jin1, Linsen Dai1, Jinbiao Zheng1and Chaoneng Ji2
1
Center of Analysis and Measurement and2State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, PRChina
The b form of human cAMP-dependent protein kinase
inhibitor (human PKIb), a novel heat-stable protein, was
isolated with high yield using a bacterial expression system
Assays of PKI activity demonstrated that purified PKIb
inhibits the catalytic subunit of cAMP-dependent protein
kinase FTIR, Raman spectroscopy and CD experiments
implied that human PKIb contained only small amounts of
a-helix and b-structures, but large amounts of random coil
and turn structures, which may explain its high thermosta-bility The details of its conformational changes in response
to heat were studied by CD experiments for the first time, revealing that the protein unfolded at high temperature and refolded when decreased to room temperature
Keywords: CD; FTIR; human PKIb; MS; Raman spectro-scopy
Signaling through cAMP-dependent protein kinase (cAPK)
is a common pathway for many cellular processes
Regu-lation of cAPK is achieved by both inhibition and
subcellular localization The best understood control
mech-anism for cAPK activity is achieved through the regulatory
(R) subunit Two catalytic (C) subunits bind to a dimer of
R subunits to yield an inactive holoenzyme Cooperative
binding of two molecules of cAMP to each R subunit causes
dissociation of the holoenzyme complex and release of
two active C subunits [1,2]
In addition, there is a second level of regulation of cAPK
activity by protein kinase inhibitors (PKIs) The PKIs are
specific and potent inhibitors of the C subunit; however,
unlike the R subunit, PKI inhibition of the C subunit is not
relieved by cAMP [3–5] Moreover, the PKIs also serve to
localize the C subunit in the cell It has been demonstrated
that C–PKI complexes are more rapidly exported out of the
nucleus than the C subunit alone and that this process is both
temperature- and ATP-dependent [6] Specifically, a nuclear
export signal has been identified on PKIs corresponding to a
leucine-rich sequence conserved in the PKIs [3,7]
To date, three mouse PKI genes encoding three isoforms
(PKIa, PKIb and PKIc) [3,8,9] and two human genes
encoding PKIa [10] and PKIc (Genbank accession numbers
AB019517 and AF182032) have been cloned A newgene
of human PKIb (Genbank accession number AF225513)
has been cloned for the first time based on the human fetal
brain cDNA library in our laboratory [11]
To explore further the structure of human PKI and understand the nature of the protein, here we report the purification and structural characteristics of human PKIb Experimental procedures
Materials Unless otherwise stated, all reagents were of analytical grade The bacterial strain Escherichia coli BL21(DE3) pLySs and plasmid vector pET were stored in our laboratory Enzymes used in vector construction were from New England Biolabs DEAE Sepharose Fast Flowexchange media were obtained from Amersham Pharmacia Biotech Company
Isolation and sequencing of a cDNA clone encoding human PKIb
A high quality cDNA library was constructed using human fetal brain poly(A+) RNA by our laboratory A full length cDNA clone encoding human PKIb was obtained by large-scale sequencing [11]
Construction of human PKIb expression vector The human PKIb coding region was amplified by PCR with oligonucleotides 5¢-CCCCATATGATGAGGACAGATT CATCAAAAATG-3¢ and 5¢-CATGGATCCTCATTTT TCTTCATTTTGAGGC-3¢ The amplified PCR fragment was inserted into plasmid vector pET between the endo-nuclease sites NdeI and HindIII The constructed expression vector was confirmed with by sequencing and found to contain no errors, and was transformed into E coli BL21 (DE3) pLySs
Production of recombinant human PKIb
A single colony was transferred to 30 mL of M9 medium [12], which was supplemented with 0.05% (w/v) thiamine,
Correspondence to C Ji, State Key Laboratory of Genetic
Engineer-ing, School of Life Science, Fudan University, Shanghai 200433,
PR China Fax: + 86 21 65642502, Tel.: + 86 21 65643958,
E-mail: chnji@fudan.edu.cn
Abbreviations: C, catalytic subunit; cAPK, cAMP-dependent protein
kinase; PKIb, b form of cAMP-dependent protein kinase inhibitor;
R, regulatory subunit.
(Received 3 December 2003, revised 21 February 2004,
accepted 12 March 2004)
Trang 2Chicago, IL, USA) The cell debris was separated from
extract by centrifugation at 25 200 g for 15 min By
addition of NaCl and NaAc (pH 5.0) to a concentration
of 0.2 molÆL)1 and 0.1 molÆL)1, respectively, the
super-natant was incubated at 85C water bath for 30 min The
denatured protein was removed by centrifugation at
25 200 g for 15 min The supernatant was precipitated
by 70% (w/v) ammonium sulfate After dialysis against
10 mM Tris/HCl (pH 8.0), the protein solution was
adjusted to pH 5.0 with acetic acid and incubated for
30 min at room temperature Precipitated proteins were
removed by centrifugation and the supernatant was then
applied to a column of DEAE Sepharose Fast Flow
(5· 25 cm) equilibrated with 5 mMsodium acetate buffer,
pH 5.0 The column was eluted with a linear gradient
(100 mL) of sodium acetate buffer, pH 5.0, from 5 to
300 mM, then from 300 to 600 mM
The eluate was monitored by absorption at 280 nm
The fraction containing the human PKIb proteins was
eluted at 350–500 mMsodium acetate buffer The pooled
fraction was stored at )20 C for further study The
concentration of the proteins was determined by the
method of Lowry et al [14] and the purity was examined
by SDS/PAGE [15]
Assay of human PKIb activity
The activity of purified human PKIb was assayed by the
inhibition of the catalytic subunit of cAPK in a 50 lL
reaction containing 0.5 units of purified catalytic subunit,
25 mM Tris/HCl (pH 7.4), 5 mM magnesium acetate,
5 mM dithiothreitol, 20 lM Kemptide and 0.1 mM
[c32P]ATP (200 cpmÆpmol)1) [13] Reactions were
incu-bated for 20 min at 30C and 25 lL aliquots spotted
onto phosphocellulose strips (Whatman ET31) The
filters were washed three times with 75 mM phosphoric
acid and radioactivity determined by scintillation
spectro-metry
MS measurement
Electrospray MS was performed on a PerkinElmer API-165
mass spectrometer equipped with Bio-Q quadrupole and
electrospray ionization source The mass-to-charge ratio
was set from 500 to 3000 with the step size of 0.25 Protein
concentration was 5.64 mgÆmL)1in HO
France), equipped with a He-Ne laser at wavelength of 632.8 nm and 6 mW of power The recorded resolution of spectrum was 1 cm)1 Protein concentration was 0.564 mgÆmL)1in H2O
CD measurements
CD spectrum was acquired on a 0.1 cm path length cell of a Jasco-715 spectrometer (Jasco, Tokyo, Japan) equipped with RTE bath/circulator (NESLAB RTE-111; NESLAB, Tokyo, Japan) After a 25 min N2purge, the spectra were recorded from 185 nm to 250 nm with a resolution of 0.2 nm and accumulated for four scans Protein concentra-tion was 0.071 mgÆmL)1in H2O
Results Cloning of the gene coding for human PKIb The cDNA clone encoding PKIb gene contains 1057 base pairs (Fig 1), and is confirmed to be a novel gene byBLAST (NCBI) analysis (Genbank accession number AF225513) This gene contains an open reading frame of 237 nucleotides and a putative polyadenylation signal ATTAAA (1018– 1023) and poly(A) tail (Fig 1) The human PKIb protein predicted by the open reading frame is 78 amino acids in length with a calculated molecular mass of 8468.2 Da and
PI of 4.69 It shows relatively low homogeneity to other known human PKI isoforms: PKIa (30%) and PKIc (23%) However, it is 74% identical to mouse PKIb1 gene
in the amino acid sequence [16] The residues of the pseudo-substrate sequence [RRNA(26–29)] which were demonstra-ted to play an important role in the high affinity for the
C subunit are conserved in human PKIb (Fig 1) [4,17,18] Likewise, a sequence highly similar to the consensus PKI nuclear export signal, LXLXLXXLXHy(45–54), (where X
is any amino acid and Hy is any hydrophobic amino acid)
is present in human PKIb (Fig 1) [3,6] Based on these elements of sequence homology, the newprotein is designated as human PKIb
Purification and characteristics of recombinant human PKIb
Protein expression and purification was applied as described above A summary of the purification procedure is given in
Trang 3Table 1 According to the activity assay, the activity was
retained above 95% (data not shown) and the recovery is
24% during the heat-treatment It implied that PKIb was a
thermostable protein and part of the protein may
copreci-pitate with other denatured proteins Starting from the 2 L
of bacterial culture, 1.52 mg of purified PKIb was obtained
with an apparent overall recovery yield of 1.2% (Table 1)
The purified PKIb show ed a single band by SDS/PAGE
(Fig 2) The assay of its activity demonstrated that the
purified PKIb inhibited the catalytic subunit of cAPK with
the specific activity of 6.0· 104unitÆmg)1(Fig 3A).The Ki
value determined from the replots was 0.173 nM(Fig 3B)
The experimental molecular mass (8468.0 Da) obtained
by electrospray MS was in complete accordance with the
theoretical mass of the human PKIb (8468.2 Da) [11] The
identification of the gene product of human PKIb excluded
it from the interference of human PKIb-70, which was
translated from another initiated site and resulted in an alternate protein with 7–8 residues less than human PKIb [11,19]
Conformation of human PKIb from FTIR, Raman and CD Absorbance spectrum and the second derivative spectrum
of FTIR for human PKIb are shown in Fig 4A and B, respectively The spectrum of the conformation-sensitive amide I¢ region (1620–1700 cm)1) exhibits five well defined
Fig 1 Nucleotide and predicted amino acid sequence of human PKIb Amino acid sequence
of human PKIb inferred from the nucleotide sequence is represented belowthe DNA sequence with the one-letter amino acid codes Nucleotide numbers are indicated at the left of the sequence The putative polyadenylation signal is underlined The pseudosubstrate sequence is marked with a rectangular frame and the nuclear export signal sequence is marked with a rounded frame.
Table 1 Typical purification of recombinant human PKIb Human
PKIb determined from electrophoresis analysis of the proteins.
Fraction
Total protein (mg)
Human PKIb (mg)
Recovery (%) Crude extract 1250 125 100
Heat-treatment 47.5 30 24
(NH 4 ) 2 SO 4
fractionation
Fig 2 Electrophoresis analysis of proteins present in fractions from bacterial cultures containing human PKIb expression vectors A, 2.5 lL
of markers; B, 5 lL of the crude extract from the cells; C, 5 lL of heat-treated crude extract; D, 5 lL of (NH 4 ) 2 SO 4 fractionation; E, 5 lL of DEAE eluate; F, 10 lL of DEAE eluate Fractions were analyzed by SDS/PAGE (12%) followed by staining with Coomassie Brilliant Blue.
An arrowindicates the position of human PKIb.
Trang 4absorption peaks at 1683, 1669, 1653, 1647 and 1636 cm)1,
and two shoulders at 1663 and 1625 cm)1, which indicate
that the amide I¢ mode consists of various overlapping
components (Fig 4A) These component bands can be
better visualized with the second derivative spectrum in
Fig 4B, which reveals, in addition to the major bands
described above, another band at 1675 cm)1 On the basis
of 21 proteins of known structure, Byler & Susi [20] have
assigned 11 well-defined frequencies in the amide I¢ region
to the secondary structural elements Hence the peak at
1653 cm)1is assigned to a-helix of human PKIb; the bands
at 1675, 1636 and 1625 cm)1can be assigned to extended
chain structures; the bands at 1683, 1669 and 1663 cm)1are
possibly assigned to turns or bends; and the band at
1647 cm)1is likely to be the random coil structure
As occurred in FTIR, the Raman amide III¢ and I¢
regions of human PKIb are sensitive to secondary structure
of protein and one might expect the above solution structure
to be reflected in Fig 5 The 1652 and 1270 cm)1 bands
can be assigned to a-helix in the human PKIb; the 1666
and 1247 cm)1 bands are the characteristic frequencies
of the random coil structure Meanwhile, the 1682 and
1255 cm)1 bands can be assigned to extended chain or
b-turn structure [21]
The quantitative contributions of the individual amide I¢ component bands, determined by band fitting of the absorbance spectrum of Fig 4A, are shown in Fig 6 and Table 2 From Table 2, the sum of individual amide I¢ intensity and the intensity percent of each peak from human
Fig 3 Activity analysis of inhibition of cAMP-dependent protein kinase
activity by inhibitor peptides (A) The inhibitory potency was assayed
through incubation, increasing concentration of human PKIb with
cAPK (B) Kinetic analysis of inhibition of cAMP-dependent protein
kinase activity by inhibitor peptides Assays were performed essentially
as described by Thomas et al [13] except that the reactions contain
20 lm, 10 lm, 5 lm, 2.5 lm Kemptide The K i value determined from
the repots was 0.173 nm.
Fig 4 FTIR Spectrum of human PKIb (A) Absorbance spectrum was measured for PKI in D 2 O and ratio determined against a single-beam background collected with no cell (B) Second derivative spectrum.
Fig 5 Raman spectrum of human PKIb in H 2 O.
Trang 5PKIb can easily be calculated According to the above peak
assignment, the relative contents of secondary structure in
the human PKIb based upon the fitted spectrum are shown
in Table 3
The CD spectrum of human PKIb at 25C (Fig 7A)
shows that the protein has a major unordered structure, as
indicated by the presence of a very strong negative band
at 198 nm The negative band near 220 nm results from
overlapping of the bands of b-sheet (215 nm) and a-helix
(209 nm and 222 nm) [22] Analysis of the solution CD spectrum of human PKIb with the computer program CONTIN (http://lamar.colostate.edu/sreeram/CDPro) also indicates that the protein has a dominant unordered structure (Table 3), which is consistent with the result obtained through infrared spectroscopy
CD spectrum of thermal unfolding and refolding
of human PKIb The conformational changes of the human PKIb were monitored by CD from 25 to 95C Four curves corres-ponding to 25, 50, 75 and 95C are shown in Fig 7A It was found that the absorbance at 198 nm increased when the temperature was increased, which implied that the human PKIb unfolded gradually When the temperature was gradually lowered from 95 to 25C (Fig 7B), the absorbance at 198 nm decreased, which means that the protein refolded again
Discussion This study described the cloning, expression, purification, identification and characterization of a member of the PKI family By cloning and construction of a high- expression
Fig 6 Amide I¢ infrared band of human PKIb in D 2 O buffer with the
best-fitted individual component band Spectrum exhibits the individual
Lorentzen components.
Table 2 Component band positions, relative integrate intensities and
secondary structure assignment for human PKIb Fractional area refers
to infrared amide I¢ band.
Infrared
Fractional area Raman
Secondary structure assignment
m (cm)1)
1683 0.104 1682 b-Turn
1675 0.077 Extended chain
1653 0.385 1652 a-Helix
1647 0.510 1666 Random coil
1636 0.379 Extended chain
1625 0.202 Extended chain
1270 a-Helix
1255 Extended chain or b-turn
1247 Random coil
Table 3 Fractional composition of secondary structure for the human
PKIb as estimated by infrared spectroscopy and CD spectroscopy.
Secondary structure Infrared amide I¢ band CD
Fig 7 CD spectrum of the human PKIb in the different temperatures (A) Increase from 25 to 95 C and (B) decrease from 95 to 25 C at 185–250 nm (for clarity of comparison, only part of the spectrum is shown).
Trang 6and turn structures, with a small amount of a-helix and
b structures However, this was compatible with previous
reports [23] that minimal structure should be maintained to
resist both high temperature and lowpH The
conforma-tional changes of protein against temperature were
evalu-ated by CD spectrum It could be the reason why human
PKIb was a heat-stable protein, as it unfolded at high
temperature and refolded when gradually descended to
room temperature This mechanism may also be applied to
other kinds of protein in the PKI family
Bacterially produced human PKIb provided a useful tool
for studies of the catalytic subunit of the cAMP-dependent
protein kinase Its ability to readily alter the PKI coding
sequence could permit further studying of human PKIb and
understanding of the function of cAPK
Acknowledgements
This study was supported by the National Natural Science Foundation
Grant of China (30070161).
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