Báo cáo khoa học: Purification and structural study of the b form of human cAMP-dependent protein kinase inhibitor pdf

Purification and structural study of the b form of human cAMP-dependent protein kinase inhibitor Rong Jin 1 , Linsen Dai 1 , Jinbiao Zheng 1 and Chaoneng Ji 2 1 Center of Analysis and Measurement and 2 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, PR China 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 new gene 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 Flow exchange 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 NdeIandHindIII. 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) Eur. J. Biochem. 271, 1768–1773 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04087.x 0.1% (w/v) D -biotin, 0.1% (w/v) folic acid, 0.1% (w/v) pyridoxal, 0.01% (w/v) riboflavin, 1 m M MgSO 4 and 0.1 mgÆL )1 ampicillin. Following growth overnight, the culture was diluted with 2 L of the same medium and cultured at 37 °C with shaking until D 600 reached 0.8–1.0. Isopropyl thio-b- D -galactoside was then added to 1 m M and the cultures were induced at 30 °C for 10 h. The cells were collected by centrifugation at 2800 g for 15 min and stored at )20 °C until use. Purification of recombinant human PKIb The cells from the 2 L culture were resuspended in 150mL of 10m M Tris/HCl (pH 8.0), 1 m M EDTA (pH 8.0) [13] and lysed by the ultrasonic homogenizer 4710 series instrument (Cole-Parmer Instrument Co., Chicago, 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 85 °C 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 m M 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 m M sodium 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 m M , then from 300 to 600 m M . The eluate was monitored by absorption at 280 nm. The fraction containing the human PKIb proteins was eluted at 350–500 m M sodium 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 m M Tris/HCl (pH 7.4), 5 m M magnesium acetate, 5m M dithiothreitol, 20 l M Kemptide and 0.1 m M [c 32 P]ATP (200 cpmÆpmol )1 ) [13]. Reactions were incu- bated for 20 min at 30 °Cand25lL aliquots spotted onto phosphocellulose strips (Whatman ET31). The filters were washed three times with 75 m M 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 )1 in H 2 O. FTIR measurements FTIR spectrum was measured using a PerkinElmer spec- trometer. To maximize signal to noise radio, 1024 scans were collected. The samples were in a PerkinElmer solution cell with CaF 2 window and spacer. Absorbance spectrum was obtained against a single beam background spectrum collected with no cell. Protein concentration was 5.64 mgÆmL )1 . The protein solutions were exchanged with D 2 O by repeated lyophilization. The final exchanged samples were dissolved in > 99.9% D 2 O. Raman measurement Raman spectrum was recorded by a Dilor-Labram 1B Raman spectrometer (Jobin Yvon Ltd, Villeneuve d’Ascq, 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 )1 in H 2 O. 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 N 2 purge, 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 )1 in H 2 O. 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 by BLAST (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 PKIb1gene 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 new protein 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 Ó FEBS 2004 Purification and structural study of human PKIb (Eur. J. Biochem. 271) 1769 Table 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 showedasinglebandbySDS/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 · 10 4 unitÆmg )1 (Fig. 3A).The K i value determined from the replots was 0.173 n M (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. Aminoacidsequence of human PKIb inferred from the nucleotide sequence is represented below the 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 6.36 4.2 3.4 DEAE 1.52 1.52 1.2 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 lLofheat- treated crude extract; D, 5 lLof(NH 4 ) 2 SO 4 fractionation; E, 5 lLof DEAEeluate;F,10lL of DEAE eluate. Fractions were analyzed by SDS/PAGE (12%) followed by staining with Coomassie Brilliant Blue. An arrow indicates the position of human PKIb. 1770 R. Jin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 absorption 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 )1 is assigned to a-helix of human PKIb; the bands at 1675, 1636 and 1625 cm )1 can be assigned to extended chain structures; the bands at 1683, 1669 and 1663 cm )1 are possibly assigned to turns or bends; and the band at 1647 cm )1 is 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 canbeassignedtoa-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]exceptthatthereactionscontain 20 lm, 10 lm, 5 lm, 2.5 lmKemptide.TheK 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. Ó FEBS 2004 Purification and structural study of human PKIb (Eur. J. Biochem. 271) 1771 PKIb 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 25 °C(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 95 °C. Four curves corres- ponding to 25, 50, 75 and 95 °C 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 25 °C(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 assignmentm (cm )1 ) 1683 0.104 1682 b-Turn 1675 0.077 Extended chain 1669 0.207 b-Turn 1663 0.238 b-Turn 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 a-Helix 0.19 0.22 Extended chain 0.31 0.29 Unordered 0.50 0.49 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 °Cat 185–250 nm (for clarity of comparison, only part of the spectrum is shown). 1772 R. Jin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 vector for human PKIb, milligrams of human PKIb could be produced conveniently and rapidly (Table 1). Heat treatment, DEAE ion exchange chromatography and gel filtration chromatography were usually the main processes for purification of PKIs [13]. In this process of purification, with the addition of NaCl and NaAc to the extract prior to heat treatment, many more contaminants were removed compared to the previous reports [4,13]. Followed by ammonium sulfate precipitation and DEAE sepharose fast flow chromatography, highly pure protein of human PKIb was readily produced. By reducing the step of gel filtration chromatography, the purification process was simplified. This method would be helpful to purify other kinds of proteins in the PKI family. The combined results of FTIR and CD showed that human PKIb contained large amounts of random coil and 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 low pH. 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. 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Purification and structural study of the b form of human cAMP-dependent protein kinase inhibitor Rong Jin 1 , Linsen Dai 1 , Jinbiao Zheng 1 and Chaoneng. 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