©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression, Characterization, and Crystallization of the Catalytic Core of the Human Insulin Receptor Protein-tyrosine Kinase Domain (*)

(Received for publication, November 16, 1994; and in revised form, January 13, 1995)

Lei Wei (1)(§) Stevan R. Hubbard (2) Wayne A. Hendrickson (2) (3) Leland Ellis (1)(¶)

From the  (1)W. M. Keck Center for Genome Informatics, Institute of Biosciences and Technology, Texas A & M University, Houston, Texas 77030 and the (2)Department of Biochemistry and Molecular Biophysics and (3)Howard Hughes Medical Institute, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The deduced primary sequence of the cytoplasmic protein-tyrosine kinase domain of the insulin receptor contains a conserved kinase homology region (receptor residues 1002-1257) flanked by a juxtamembrane region and a C-terminal tail. A soluble 48-kDa derivative (residues 959-1355) containing these regions but lacking the first six residues of the juxtamembrane region had earlier been synthesized in Sf9 cells using a baculovirus expression system. The catalytic core of the kinase domain was studied first by proteolytic analysis of the 48-kDa kinase and then by expressing a series of truncated kinase domains in transiently transfected COS cells. Based on these studies, two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, were overexpressed in Sf9 cells. Biochemical characterization of the 35-kDa kinase revealed that the core kinase conserved the major functional properties of the native receptor kinase domain. Activity of the 35-kDa kinase toward a synthetic peptide increased more than 200-fold upon autophosphorylation, which occurred exclusively at Tyr-1158, Tyr-1162, and Tyr-1163; the largest increase was observed between bis- and trisphosphorylation of the kinase. The activated 35- and 48-kDa kinases were similar with respect to specific activity and ATP and Mg requirements for peptide phosphorylation. Moreover, autophosphorylation appeared to initiate predominantly at Tyr-1162, immediately followed by phosphorylation at Tyr-1158 and then at Tyr-1163. The rate of autophosphorylation was dependent on enzyme concentration, consistent with a trans-phosphorylation mechanism. Finally, the 35-kDa kinase was crystallized, making possible elucidation of its three-dimensional structure by x-ray crystallography.


INTRODUCTION

The insulin receptor is a disulfide-linked heterotetrameric transmembrane glycoprotein composed of two extracellular alpha-subunits and two beta-subunits that contain a single trans-membrane domain and a cytoplasmic protein-tyrosine kinase domain(1, 2) . The binding of insulin to the alpha-subunit of the receptor on intact cells immediately activates the proteintyrosine kinase activity in the beta-subunit, resulting in rapid autophosphorylation of multiple tyrosine residues: Tyr-1158, Tyr-1162, and Tyr-1163 in the kinase homology region; Tyr-1328 and Tyr-1334 in the C-terminal tail; and one or more of Tyr-965, Tyr-972, and Tyr-984 in the juxtamembrane region (3, 4, 5) . Autophosphorylation of the three core tyrosines (1158, 1162, and 1163) results in activation of the kinase toward exogenous substrates(5, 6, 7) . Substantial evidence has accumulated suggesting that the insulin-stimulated protein-tyrosine kinase activity of the insulin receptor is essential for mediation of insulin action(8, 9, 10, 11) . Insight into the structure and function of the kinase domain is thus critical for understanding insulin action.

The cytoplasmic protein-tyrosine kinase domain of the insulin receptor has been expressed as an active soluble kinase in both stably transfected mammalian Chinese hamster ovary cells (12) and insect Sf9 cells(13, 14) , demonstrating that the cytoplasmic kinase domain is capable of autonomous function. Furthermore, a soluble 48-kDa derivative, which contains the cytoplasmic domain lacking the first six residues after the transmembrane domain (residues 959-1355), has been shown to exhibit the major functional properties of the native receptor kinase(13, 15, 16) . Overproduction of this soluble enzyme has made possible structure and function studies of the kinase domain by biochemical as well as biophysical approaches including NMR spectroscopy(17, 18, 19) .

However, despite the increasing body of data derived from in vitro studies of the soluble kinase, the kinase domain is poorly characterized in structural terms. The 48-kDa kinase has been found to be resistant to crystallization, most likely due to the presence of unstructured segments in the molecule. (^1)Furthermore, no crystallographic structure of a protein-tyrosine kinase has been determined that could be used to construct, by homology, a model of the insulin receptor kinase. In fact, the four protein kinases for which the three-dimensional structures have been reported all belong to the family of protein serine/threonine kinases (cAMP-dependent kinase, cyclin-dependent kinase 2, the mitogen-activated protein kinase Erk2, and twitchin kinase) ((20) -23, reviewed in (24) ). Engineering new derivatives of the insulin receptor kinase domain suitable for x-ray analysis is thus required to determine the three-dimensional structure of this kinase domain, which is of fundamental importance for understanding its mechanism of catalysis.

In the present study, we determined the catalytic core of the kinase domain first by proteolytic analysis of the 48-kDa kinase and then by expressing truncated kinase domains in tran-siently transfected COS cells. The results derived from these experiments provide critical information concerning the subdomain organization of the kinase domain. To produce large quantities of the core kinases, we have expressed two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, using the baculovirus expression system. In addition, biochemical characterization of the 35-kDa kinase reveals that the core kinase exhibits functional properties characteristic of those of the native receptor kinase. Finally, the 35-kDa kinase was found to be appropriate for crystallization and x-ray analysis.


MATERIALS AND METHODS

Proteolytic Analysis of the 48-kDa Kinase

Proteolytic experiments were performed for a fixed time as a function of proteolytic enzyme concentration rather than as a time course at fixed enzyme concentration. In a typical experiment, 3-fold serial dilutions (six) of a stock solution (1 mg/ml) of the proteolytic enzyme were prepared. A reaction mixture of 4 µl included 1 µl of 3 mg/ml purified or partially purified 48-kDa kinase, 1 µl of 200 mM sodium phosphate, pH 7.6, 1 µl of the proteolytic enzyme dilution, and 1 µl of water. The reactions were run for 1 h at room temperature and stopped by the addition of 4 µl of Laemmli sample buffer followed by immediate boiling. Samples were analyzed by SDS-PAGE (^2)on 10-15% gradient PhastGels (Pharmacia Biotech Inc.).

Construction of COS Cell Expression Plasmids for Truncated Soluble Kinases

N-terminal Deletion

The starting construct was the plasmid peiBgl(12) , which comprises a human insulin receptor cDNA cloned into the expression vector pECE (11) containing the SV40 early promoter. This plasmid encodes the 48-kDa soluble kinase that contains two heterologous residues (Met-Asp) followed by receptor residues 959-1355. The plasmid peiBgl was digested with HindIII (in the 5` polylinker) and XhoI (bp 3195 of the receptor cDNA), and the resulting 4.1-kbp fragment (bp 3159-4443 of the cDNA plus the pECE vector) was ligated to a double-stranded synthetic oligonucleotide including 5` HindIII and 3` XhoI overhangs, a consensus Kozak sequence for the initiation of translation with an NcoI site (underlined sequence) at the ATG initiation codon, and the cDNA sequence extending the 5` end of the cDNA insert to the codon coding for Asp-977, viz.

Three other double-stranded synthetic oligonucleotides extend the 5` end of the cDNA insert to the codons coding for Val-983, Val-985, and Val-991, respectively. The resulting plasmids, designated peDeltaNT24, peDeltaNT30, peDeltaNT32, and peDeltaNT38, encode the four N-terminally truncated kinase domains lacking 24, 30, 32, and 38 N-terminal residues of the cytoplasmic domain, respectively (see Table 1). The plasmid peDeltaNT24 contains an additional substitution of Tyr-984 with Phe. The cDNA 5` end regions of these plasmids containing the sequences shown above were confirmed by DNA sequencing.



C-terminal Deletion

A 1.4-kbp cDNA fragment (bp 3087-4443) was isolated from the plasmid peiBgl by digestion with HindIII (in the 5` polylinker) and XbaI (in the 3` polylinker) and was subcloned into an M13 vector. A stop codon (TAA) was introduced into the cDNA at a

position corresponding to Ala-1284 or Asp-1265 using an oligonucleotide-directed mutagenesis system (Bio-Rad). Mutant cDNAs were screened by sequencing of the mutated regions. The BstXI/XbaI (bp 3439-4443) fragment containing either the Ala-1284 or Asp-1265 mutation in M13 was sequenced to ensure that no other mutation had occurred and then isolated from M13 to replace the corresponding fragment in peiBgl. The resulting plasmids, designated peDeltaCT72 and peDeltaCT91, encode receptor residues Gly-959 to Lys-1283 and Gly-959 to Lys-1264, respectively. These expression plasmids were characterized by restriction mapping and sequencing of the mutated region to ensure that the mutation had been introduced correctly.

N- and C-terminal Deletions

To provide both N- and C-terminal deletions, the BstXI/XbaI fragment (bp 3439-4443) containing the Ala-1284 mutation was isolated from the plasmid peDeltaCT72 and used to replace the corresponding part of the receptor cDNA in peDeltaNT24 and peDeltaNT32, generating peDeltaNT24CT72 and peDeltaNT32CT72, respectively. The N- and C-terminal coding sequences of the cDNA in these expression plasmids were confirmed by DNA sequencing.

Expression in COS Cells and Analysis of Recombinant Proteins

Expression in COS Cells

Transfection of COS cells with expression plasmids and extraction of transfected cells were as described(25) .

Immunoblot Analysis

The cell lysates were loaded directly on 12% SDS gel (Bio-Rad minigels) ((26) ). The proteins were transferred to Immobilon membrane (Millipore). Protein bands corresponding to recombinant soluble kinases were detected with a rabbit polyclonal antibody, designated 766, raised against human placental insulin receptors and affinity purified on an Affi-Gel column possessing bound 48-kDa kinase (a gift from Dr. R. A. Roth of Stanford University). Bound rabbit antibody on the membrane was detected with an anti-rabbit alkaline phosphatase conjugate and a chromogenic substrate (Promega).

Tyrosine Kinase Assay

Recombinant proteins were immunoprecipitated from cell lysates with antibody 766 as described (25) . The immune complex was incubated in the reaction buffer including 50 mM Hepes, pH 7.5, 2 mM dithiothreitol, 4 mM MgCl(2), 4 mM MnCl(2), and 100 µg/ml bovine serum albumin for 10 min at 30 °C. 2 µCi of [-P]ATP (final concentration, 0.25 mM) was then added. After incubation for 10 min, reactions were terminated by adding Laemmli sample buffer followed by electrophoresis on SDS-PAGE and then autoradiography. 1 mM synthetic peptide substrate RRDIFETDYFRK (FYF) (17) was included in some reactions to determine phosphorylation of exogenous substrate as described (25) .

Construction of Recombinant Baculovirus Transfer Plasmids and Isolation of Recombinant Virus

The plasmid peDeltaCT72 described above was digested with XhoI (bp 3195 of the receptor cDNA) and XbaI (in the 3` polylinker) to provide a 1.2-kbp cDNA fragment. The subcloning of this fragment into the BamHI/XbaI-digested baculoviral transfer vector pVL1393 (Pharmingen) was performed with a double-stranded oligonucleotide linker with 5` BamHI and 3` XhoI overhangs complementary to BamHI of the pVL1393 vector and XhoI of the receptor cDNA fragment, respectively. The oligonucleotide linker also provides the 5` end to the cDNA insert with an NcoI site (underlined sequence) at the ATG translation initiation codon, viz.

The resulting plasmid, designated pVLDeltaNT25CT72, contains a truncated receptor cDNA, which encodes receptor residues Val-978 to Lys-1283 with the additional substitutions of Cys-981 with Ser and Tyr-984 with Phe, and is placed under the transcriptional control of the polyhedrin promoter.

The plasmid pVLDeltaNT32CT72, which encodes receptor residues Val-985 to Lys-1283, was constructed by replacing the NcoI/XbaI cDNA insert in the baculovirus transfer plasmid pVLDeltaNT25CT72 with the corresponding fragment isolated from the expression plasmid peDeltaNT32CT72 described above.

Transfer of the receptor cDNA from the recombinant baculoviral transfer plasmid to the baculovirus Autographa californica nuclear polyhedrosis virus DNA was performed by homologous recombination into the native polyhedrin gene after calcium phosphate cotransfection into insect Sf9 cells with a modified baculovirus DNA, which allows one to achieve almost 100% recombination efficiencies (BaculoGold transfection kit, Pharmingen). Sf9 cells were generously provided by Dr. Max Summers (Texas A & M University) and were maintained as described(27) . After transfection, recombinant viruses were isolated by plaque assay(27) . Six independent plaques were screened for recombinant protein expression by immunoblot and the kinase activity assay described above. They all exhibited similar recombinant protein expression levels. Two recombinant viruses thus obtained, AcDeltaNT25CT72 and AcDeltaNT32CT72, were used for the studies described herein.

Purification and Characterization of the Soluble Kinases

The purification of the soluble kinases expressed in Sf9 cells was performed on a 8-ml MonoQ FPLC column followed by gel filtration chromatography on a Superdex 200 prep grade column (Pharmacia)(13, 15) . Each step of the purification was monitored by UV absorption at 280 nm, exogenous kinase activity assay, SDS-PAGE followed by Coomassie Blue staining, or immunoblot analysis. Protein was measured by the method of Bradford (42) using the Bio-Rad protein reagent and also by absorbance at 280 nm with an estimated extinction coefficient of 39,170 M cm for the core kinases and 45,810 M cm for the 48-kDa kinase; the latter value was confirmed by amino acid analysis of the 48-kDa kinase.

Autophosphorylation reactions included 50 mM Hepes, pH 7.5, 2 mM dithiothreitol, MgCl(2) or MnCl(2), and ATP at indicated concentrations. Reactions were terminated by adding Laemmli sample buffer or by adding EDTA to 20 mM, and phosphoproteins were separated by SDS-PAGE or by native PAGE (Bio-Rad minigels; all the procedures are identical to those of SDS-PAGE, except that SDS is not included in buffers and the gels are run at 4 °C).

To determine the autophosphorylation sites of the soluble kinases, the P-labeled kinases were excised from the untreated wet gel, electroeluted from the gel, digested with tosylphenylalanyl chloromethyl ketone-trypsin, and subjected to either phosphoamino acid analysis or two-dimensional phosphopeptide mapping as described(28) .

Exogenous kinase activity assays were performed with 50 mM Hepes, pH 7.5, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, MgCl(2) and ATP at indicated concentrations, and 1 mM FYF. Initial velocities were measured during the first 10% of substrate phosphorylation.

Crystallization of the 35-kDa Kinase

The 35-kDa kinase used for crystallization was purified on Q-Sepharose, Superdex 200, and MonoQ columns. Crystals were grown by vapor diffusion in hanging drops in 24-well Linbro plates (Flow Laboratories). Polyethylene glycol (PEG) 6000 was obtained from Fluka. A 2 M stock solution of the malate/imidazole buffer used in crystallizations was prepared by mixing 2 M solutions of malic acid and imidazole to the desired pH. Streak seeding was performed with a glass fiber, and an Eppendorf Pipetman was used for transferring seed crystals between various macroseeding solutions.


RESULTS AND DISCUSSION

Determination of the Catalytic Core

The major structural feature deduced from the primary sequence of the cytoplasmic domain of the insulin receptor is a conserved kinase homology region (residues 1002-1257) that is flanked by a juxtamembrane region (residues 953-1001) and a C-terminal tail (residues 1258-1355)(1, 2) . The 48-kDa kinase contains all three regions but lacks the first six N-terminal residues of the cytoplasmic domain. Because attempts to crystallize this soluble derivative were unsuccessful, most likely due to the presence of unstructured fragments, we have undertaken limited proteolytic analysis and then deletion analysis of the 48-kDa kinase to determine the minimal structural requirements for a functional core kinase.

Proteolytic Analysis

Treatment of the 48-kDa kinase with trypsin results in a deletion of 10 kDa from the C-terminal end of the protein as determined by N-terminal sequencing. It should be noted that there are no Arg or Lys residues in the juxtamembrane region. The tryptic cleavage site in the C-terminal end was not determined, but the observed molecular mass of the tryptic product (38 kDa) suggests that the cleavage occurs on the C-terminal side of Lys-1283.

A number of other proteolytic enzymes including papain, elastase, and subtilisin were tested for their ability to generate a stable, active kinase fragment. Elastase treatment generated the smallest active fragment with a molecular mass of 35 kDa. The N-terminal residue was identified by gas phase protein microsequencing as Ser-982. Furthermore, the sequence of peptides generated by treatment of the 35-kDa fragment with cyanogen bromide and trypsin revealed that the C terminus of this elastase-generated kinase derivative was between Glu-1286 and Glu-1291.

These proteolytic studies identified a catalytic core within the kinase domain of the insulin receptor that is flanked on both sides by protease-sensitive regions. With the aim of generating a core kinase, we first determined the N- and C-terminal boundaries of the catalytic core of the kinase domain by expressing the N- or C-terminally truncated kinase domains in COS cells. The N- and C-terminal deletions that did not result in the loss of protein stability and kinase activity were then selected to design final core kinases. Table 1summarizes the procedure and results.

N-terminal Boundary

To introduce N-terminal deletions to the cytoplasmic protein-tyrosine kinase domain, a series of oligonucleotides encoding different N-terminal sequences were linked with a receptor cDNA fragment (see ``Materials and Methods''). These N-terminally truncated kinase domains, designated DeltaNT24, DeltaNT30, DeltaNT32, and DeltaNT38, lack the 24, 30, 32, or 38 N-terminal residues of the cytoplasmic domain, respectively. All these deletions occur in the juxtamembrane region. In the DeltaNT24 kinase, the initiator codon Met is followed by Asp-977, which is the last residue of exon 16. The 30- and 32-residue deletions occur before Val-983 and Val-985, respectively, which are close to the major elastase cleavage site (before Ser-982). The 38-residue deletion is the largest deletion that can be designed using this approach. Furthermore, these N-terminal deletions combined with the point mutation, Tyr-984 to Phe, remove two (Tyr-965 and Tyr-972 for DeltaNT30) or three (Tyr-965, Tyr-972, and Tyr-984 for DeltaNT24, DeltaNT32, and DeltaNT38) tyrosines in the juxtamembrane region, one or two of which are sites of autophosphorylation(29, 30) .

To assess the expression of truncated kinase domains in transiently transfected COS cells, total cell lysates were examined by SDS-PAGE followed by Western blot analysis using antibody 766 directed against the 48-kDa soluble kinase. As illustrated in Fig. 1A, a specific band was observed in cells transfected with each of the expression plasmids encoding the N-terminally truncated kinase domains as well as for the 48-kDa kinase but not in non-transfected cells. This result indicates that these four truncated kinase domains are all expressed as a soluble protein in the cytoplasm of transfected cells (Table 1).


Figure 1: Expression analysis of the N- or C-terminally truncated kinase domains in transfected COS cells. Non-transfected COS cells (COS), transfected COS cells that transiently express the 48-kDa kinase (48k), and N-terminally (DeltaNT) or C-terminally (DeltaCT) truncated kinase domains were analyzed as follows (see ``Materials and Methods'' for detailed description). The truncated kinase domains lack 24 (Delta24), 30 (Delta30), 32 (Delta32), or 38 (Delta38) residues from the N terminus or 72 (Delta72) or 91 (Delta91) residues from the C terminus of the cytoplasmic domain. A, immunoblot analysis. Cell extracts containing 50 µg of proteins were loaded on 12% SDS gel followed by immunoblotting with antibody 766 directed against the 48-kDa soluble kinase. The positions of the molecular mass markers (in kDa) in this and the following figures are indicated on the left. B, autophosphorylation assay. Recombinant proteins were immunoprecipitated from cell extracts and were incubated with [-P]ATP followed by SDS-PAGE and autoradiography.



To determine whether these expressed truncated kinase domains are active, cell lysates were immunoprecipitated with antibody 766 and then incubated with [-P]ATP (see ``Materials and Methods''). As shown in Fig. 1B, in the case of the three truncated kinase domains, DeltaNT24, DeltaNT30, and DeltaNT32 as well as the 48-kDa kinase, a single phosphoprotein band was observed that was not visible for the largest N-terminally deleted kinase domain, DeltaNT38, as for the control immune complex with non-transfected COS cells. Furthermore, inclusion of the synthetic peptide FYF in phosphorylation reactions resulted in the incorporation of P into this peptide for the three autophosphorylated truncated kinase domains as well as for the 48-kDa kinase (data not shown). Again, the DeltaNT38 kinase had no detectable exogenous kinase activity. These results indicate that the deletion of 24, 30, or 32 residues from the N terminus of the cytoplasmic domain results in an active soluble kinase, whereas the deletion of 38 residues completely inactivates the kinase domain.

We cannot rule out the possibility that the polyclonal antibody 766 inhibits the kinase activity of the DeltaNT38 kinase in the immune complex. However, this antibody does not inhibit the kinase activity of all other truncated kinase domains, particularly the DeltaNT32 kinase, which contains only six additional N-terminal residues, suggesting that the inactivation is an intrinsic property of the DeltaNT38 kinase and that deletion of these six residues disrupts a critical structural feature of the kinase domain that is outside of the kinase homology region.

C-terminal Boundary

The deduced primary sequence of the kinase domain suggests two potential tryptic cleavage sites, Lys-1264 and Lys-1283, in the C-terminal tail of the cytoplasmic protein-tyrosine kinase domain. Lys-1264 and Lys-1283 are both C-terminal to Leu-1263, which is predicted to demarcate the C-terminal boundary of the kinase homology region of the insulin receptor, based on the deletion analysis of the src tyrosine kinase(31) . To determine whether deletion of 91 or 72 residues following Lys-1264 or Lys-1283 results in an active truncated kinase domain, a stop codon following either of these two residues was introduced into the cDNA encoding the 48-kDa kinase by site-directed mutagenesis. Expression studies of these two C-terminally truncated kinase domains, designated DeltaCT72 and DeltaCT91, in transfected COS cells were carried out as for the N-terminally truncated kinase domains described above.

Immunoblot analysis detected a specific band for the DeltaCT72 kinase with an apparent molecular mass of 38 kDa (Fig. 1A), and this expressed protein was found to display both autophosphorylation (Fig. 1B) and exogenous kinase activities (data not shown), indicating that the DeltaCT72 kinase is expressed as a soluble active kinase in COS cells. In contrast, expression of the DeltaCT91 kinase in COS cells was hardly detectable by immunoblot (Fig. 1A), and the autophosphorylation assay following immunoprecipitation failed to detect any phosphoprotein in this case (Fig. 1B), suggesting that this truncated kinase domain expressed in COS cells is unstable. Thus, the limited tryptic proteolysis of the 48-kDa soluble kinase most probably occurs after Lys-1283, and this deletion (72 residues) is larger than the largest C-terminal deletion (69 residues) so far reported (32) that does not result in the loss of protein stability and kinase activity of the protein-tyrosine kinase domain.

Core Kinases

To examine the consequence of the N-terminal deletion in combination with the C-terminal deletion on the stability and kinase activity of the kinase domain, two truncated kinase domains lacking 24 or 32 N-terminal receptor residues and 72 C-terminal residues, designated DeltaNT24CT72 and DeltaNT32CT72, were constructed and expressed in COS cells. Because immunoblot analysis of non-transfected COS cell lysates revealed a protein band migrating at 34 kDa, which is close to that estimated for the two core kinases (35 kDa for DeltaNT24CT72 and 34 kDa for DeltaNT32CT72), the lysates of transfected COS cells were separated by MonoQ FPLC before assaying kinase activity (Fig. 2). Fractions displaying kinase activity were then analyzed by immunoblotting, which revealed a recombinant protein with the expected molecular mass for each core kinase (Fig. 3A, lanes4 and 5), indicating that these two core kinases are both stable and active when expressed in COS cells. To facilitate subsequent biochemical and biophysical characterizations of the enzymes, we next attempted to express the core kinases using the baculoviral expression system in insect cells, as it was found to be very efficient for the high level expression of the 48-kDa kinase(13) .


Figure 2: Expression analysis of the soluble core kinases in transfected COS cells. The soluble cell extracts of transfected COS cells that transiently express the 48-kDa kinase (bullet) or one of the two core kinases DeltaNT24CT72 (circle) and DeltaNT32CT72 () were separated by MonoQ FPLC followed by exogenous kinase activity assay performed on each fraction as described under ``Materials and Methods.'' The activity is expressed as radioactivity incorporated into the exogenous substrate FYF.




Figure 3: Expression analysis of the soluble core kinases in Sf9 cells infected with recombinant baculovirus. A, immunoblot analysis of the soluble cell extracts. Cell extracts (100 µg of proteins) of recombinant virus-infected Sf9 cells expressing one of the two core kinases, DeltaNT25CT72 (lane 1) and DeltaNT32CT72 (lane 2), or of wild-type virus-infected Sf9 cells (lane 3) were loaded on 12% SDS gel followed by immunoblotting with antibody 766. Lanes 4 and 5 show the MonoQ FPLC-purified core kinases DeltaNT24CT72 and DeltaNT32CT72 (200 ng of protein) expressed in transfected COS cells. The positions of the core kinases (in kDa) are indicated on the right. B, Coomassie Blue-stained SDS-PAGE of the purified soluble kinases. Purification of the 48-kDa kinase (lane 1) and the two core kinases from infected Sf9 cells, DeltaNT25CT72 (lane 2) and DeltaNT32CT72 (lane 3), was carried out as described under ``Materials and Methods.'' A protein sample of 2 µg for each kinase was subjected to SDS-PAGE in a 12% gel. The positions of the soluble kinases (in kDa) are indicated on the right.



Large Scale Expression of the Core Kinases in Insect Cells

The baculovirus transfer vector pVL1393 was used to express the core kinases in insect cells as a nonfusion protein under the strong polyhedrin promoter of baculovirus A. californica nuclear polyhedrosis virus. Two transfer plasmids, pVLDeltaNT25CT72 and pVLDeltaNT32CT72, were constructed, each containing a cDNA fragment encoding a truncated kinase domain, designated DeltaNT25CT72 or DeltaNT32CT72. Compared with the core kinase DeltaNT24CT72 described above, DeltaNT25CT72 lacks the 25 (instead of 24) N-terminal residues of the cytoplasmic domain and contains one additional substitution (Cys-981 to Ser), because Cys-981 was suspected to be responsible for the slow dimerization of the 48-kDa kinase. (^3)For the other core kinase, DeltaNT32CT72, the sequence is identical to that expressed in COS cells described above.

These two transfer plasmids were then used to obtain two recombinant viruses, AcDeltaNT25CT72 and AcDeltaNT32CT72 (see ``Materials and Methods''). A recombinant protein of 35 or 34 kDa was specifically recognized by antibody 766 in cells infected with AcDeltaNT25CT72 or AcDeltaNT32CT72, respectively, but not in cells infected with wild-type virus (Fig. 3A). Exogenous substrate phosphorylation was also detectable in crude cell extracts of infected cells (data not shown), indicating that the core kinases are both stable and active when expressed in both insect and COS cells. The maximal expression level of the core kinases in Sf9 cells was found at 64 and 72 h postinfection, instead of at 42 h as for the 48-kDa kinase.

Purification of the core kinases from infected cells was carried out on a MonoQ anion exchange FPLC column followed by gel filtration chromatography (see ``Materials and Methods''). The final purified sample migrated as a single band of apparent molecular mass of 35 (DeltaNT25CT72) or 34 (DeltaNT32CT72) kDa on SDS-PAGE (Fig. 3B). The purity of the sample was estimated to be 95% based upon densitometry scanning of Coomassie Blue-stained gels. On gel chromatography, the core kinases elute at positions of 38 (DeltaNT25CT72) and 36 (DeltaNT32CT72) kDa between ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa) (data not shown), indicating that the core kinases are monomeric enzymes. The purified core kinases can be stored at 4 °C for several months without loss of activity, whereas the 48-kDa kinase slowly undergoes degradation. The recombinant proteins account for 2-5% of the total cell proteins in infected cells. This high level of expression combined with the two-step purification procedure resulted in an overall yield of about 1 mg of purified enzyme for a typical preparation of 200 ml of culture (2 times 10^6 cells/ml).

Characterization of the Core Kinases

There are three major differences between the core kinases, DeltaNT25CT72 and DeltaNT32CT72 (named 35- and 34-kDa kinases, respectively), and the 48-kDa kinase: 1) the stability of the core kinases is significantly greater, thus facilitating the production of large quantities of the enzyme and subsequent characterizations; 2) the core kinases are significantly smaller (Table 1) and lack N- and C-terminal protease-sensitive segments outside of the catalytic core and thus may be suitable for protein crystallization; and 3) the core kinases contain only three (Tyr-1158, Tyr-1162, and Tyr-1163) of the six to eight autophosphorylation sites present in the 48-kDa kinase, making them attractive for the study of phosphorylation kinetics.

For studies of the core kinases to be relevant to the kinase in the native receptor, the core kinases must exhibit major functional properties of the native receptor kinase. The most important characteristic of the insulin receptor kinase is the activation of its catalytic activity toward exogenous substrates by autophosphorylation of the three core tyrosines of the kinase(3, 4, 5, 6, 7) . To examine whether the core kinases preserve these functional properties, we have investigated the correlation between the autophosphorylation state of the enzyme and its activation toward peptide substrate FYF and have determined the autophosphorylation sites. Furthermore, we have also examined the order and the mechanism of autophosphorylation at the three core tyrosines. Most of the enzymatic characterizations were performed on the 35-kDa kinase.

Activation by Autophosphorylation

Insulin receptor protein-tyrosine kinase requires divalent metal ions for its catalytic activity. Because high ATP concentrations are required for maximal activation of the kinase by Mg(33) , we have chosen 4 mM ATP and 8 mM Mg for autophosphorylation assays of purified 35-kDa kinase. The time course of autophosphorylation is shown in Fig. 4. Within 10 min, about 2 mol of phosphate were incorporated per mol of enzyme, and a steady state of 2.8 mol of phosphate/molecule of enzyme was reached after 25-30 min of incubation. The specific enzyme activity of the core kinase was measured with 1 mM FYF, 0.25 mM ATP, and 8 mM Mg. Autophosphorylation during substrate incubation was undetectable because the high substrate concentration (1 mM) considerably inhibits autophosphorylation of the kinase and the low ATP concentration (0.25 mM) greatly reduces the rate of autophosphorylation even in the absence of the substrate (data not shown). The specific activity of the core kinase is 1.5 nmol/min/nmol in its unphosphorylated form, compared with 363 nmol/min/nmol for the most highly phosphorylated state, indicating a more than 200-fold increase in activity due to autophosphorylation. Thus, like the intact insulin receptor, the core kinase is activated by autophosphorylation.


Figure 4: Activation by autophosphorylation of the 35-kDa kinase. Enzyme (2 µM) was incubated in the presence of 4 mM [-P]ATP and 8 mM MgCl(2). At the indicated times, aliquots of reactions were terminated by addition of 20 mM EDTA. The P incorporated into the kinase (bullet) was determined after SDS-PAGE in a 12% gel followed by scintillation counting of the Coomassie Blue-stained bands. The exogenous kinase activity (circle) was measured with diluted aliquots in a 10-min assay with 1 mM FYF, 0.25 mM [-P]ATP, and 8 mM MgCl(2). Stoichiometry of phosphorylation of the kinase was calculated from the specific activity of the [-P]ATP and the amount of the kinase present in each sample. The exogenous kinase activity is expressed as the percentage of the measured maximal activity, which was achieved after 25 min of autophosphorylation.



Because several phosphate molecules were incorporated into the core kinase during the course of activation, we attempted to separate the multiple forms of phosphorylated enzyme by native PAGE, which separates the proteins by their charge as well as by their size. As illustrated in Fig. 5A, the unphosphorylated kinase migrated as a single major band, and three additional bands, apparently containing one, two, or three phosphate groups, appeared progressively during the course of autophosphorylation. The unphosphorylated kinase also displayed a minor band under the major band. This minor band, representing less than 10% of the unphosphorylated kinase, disappeared upon autophosphorylation and was not detectable on SDS-PAGE, suggesting that the minor band results from a conformational change or from covalent modifications that modify the charge of the molecule. The three phosphorylated forms, designated 35-kDa pY1, 35-kDa pY2, and 35-kDa pY3, correspond in fact to the mono-, bis-, and trisphosphorylated kinases on the three core tyrosines (see below).


Figure 5: Native PAGE analysis of the autophosphorylation cascade of the 35-kDa kinase and correlation with enzyme activity. A, autophosphorylation of the 35-kDa kinase was carried out as described in the legend of Fig. 4, and, at indicated times, aliquots of each reaction were analyzed by 7.5% native PAGE followed by Coomassie Blue staining. B, correlation between the exogenous kinase activity and mono-, bis-, and trisphosphorylations of the 35-kDa kinase. Exogenous kinase activity (circle) was measured as described in Fig. 4and is expressed as the percentage of the measured maximal activity. The quantities of monophosphorylated (bullet), bisphosphorylated (), and trisphosphorylated () forms were determined by densitometry scanning or by Cerenkov counting of the bands (35-kDa pY1, 35-kDa pY2, and 35-kDa pY3; both methods yielded similar results) and are expressed as the percentage of the amount of the kinase present in each sample (sum of all forms including the unphosphorylated form).



Fig. 5B shows the kinetic correlation between the extent of exogenous kinase activity and the extent of each of the three phosphorylated forms. Within the first 10 min of autophosphorylation, 35-kDa pY1 and 35-kDa pY2 accumulate, which is associated with a slight increase in the kinase activity (20% of maximal activation). The trisphosphorylated form, 35-kDa pY3, appears at 10 min and becomes a major form after 20 min. About 80% of maximal activation was observed after 10-25 min of autophosphorylation, indicating that the largest increase in the kinase activity occurs between bis- and trisphosphorylation of the kinase. The activities of 35-kDa pY1 and 35-kDa pY2 are estimated to be 2 and 10% of that of 35-kDa pY3, respectively. This result is consistent with previous studies showing that trisphosphorylation of the kinase domain is necessary for complete activation of the insulin receptor(5, 6, 34) .

We then compared catalytic properties of the core kinase to those of the 48-kDa kinase. Previous studies of the 48-kDa kinase indicate that the extent of activation following autophosphorylation depends on the activation conditions as well as on the substrate and the conditions for substrate phosphorylation(15, 19) . Under the activation conditions described above, about 5 mol of phosphate were incorporated per mol of the 48-kDa kinase at the maximal activation state, which was reached after 25-30 min of incubation. The extent of activation (200-fold) as well as the specific activity at maximal activation state (340 nmol/min/nmol) were found to be similar to those of the core kinase. Requirements for ATP and Mg of the 35- and 48-kDa kinases were determined with the activated enzyme (pre-autophosphorylated for 30 min under the conditions described above) and 1 mM FYF. The K(m) for ATP of the core kinase, determined with 8 mM Mg, is 189 µM, which is similar to that of the 48-kDa kinase (183 µM). The Mg activation profiles, determined with 0.25 mM ATP, are also similar for the 35- and 48-kDa kinases with half-maximal activation of the kinase activity around 1.5 mM and maximal activation achieved at 8 mM. These observations indicate that the 35-kDa kinase displays similar catalytic properties to the 48-kDa kinase.

Autophosphorylation Sites

When the P-labeled 35-kDa kinase was analyzed for phosphoamino acid content, the phosphorylation was found exclusively on tyrosine residues (data not shown). Native PAGE analysis of the phosphorylated 35-kDa kinase indicated the presence of three phosphorylated forms, 35-kDa pY1, 35-kDa pY2, and 35-kDa pY3, which progressively appeared at 2, 5, and 10 min, respectively (Fig. 5A). The phosphorylated kinases at these time points were analyzed by two-dimensional tryptic phosphopeptide mapping (Fig. 6), which resolves the three core tyrosine autophosphorylation sites as a family of six phosphopeptides (peptides C0, C1, B2, B3, A1, and A2)(28, 35) . The appearance of monophosphorylated (C0 and C1), bis-phosphorylated (B2 and B3), and trisphosphorylated (A1 and A2) core peptides (Fig. 6) correlates with that of 35-kDa pY1, 35-kDa pY2, and 35-kDa pY3 (Fig. 5A), indicating that these three forms correspond to the mono-, bis-, and trisphosphorylated kinases on the three core tyrosines. The monophosphopeptide C0, which represents less than 10% of the monophosphorylated species, has not been reported for two-dimensional tryptic phosphopeptide maps of the autophosphorylated human insulin receptor (28) and the 48-kDa kinase(16) . The identification of this peptide on phosphopeptide maps was determined using the synthetic peptide DIYETDY(P)YRK(35) .


Figure 6: Two-dimensional tryptic phosphopeptide analysis of the autophosphorylation cascade of the 35-kDa kinase. Autophosphorylation was carried out as described in the legend of Fig. 4. P-Labeled kinases at 2 (2`), 5 (5`), 10 (10`), and 90 (90`) min were electroeluted from SDS-PAGE, digested with trypsin, and then subjected to two-dimensional thin-layer chromatography as described under ``Materials and Methods.'' The small circles indicate the sample origin and the large circles represent the dinitrophenyl lysine standard. The drawing in the top panel is a key identifying the observed phosphopeptides. Peptides C0, C1, B2, B3, A1, and A2 are related peptides containing Tyr-1158, Tyr-1162, and Tyr-1163 phosphorylated on one (C0 and C1), two (B2 and B3), or three (A1 and A2) tyrosine residues. The sequence is DIYETDYYR for C1, B3, and A2 or DIYETDYYRK for C0, B2, and A1(35) .



It should be noted that the bisphosphorylated form is not completely converted into the trisphosphorylated form, as 35-kDa pY2 represents 20% of the kinase for the most highly phosphorylated state (Fig. 5B). Prolonged incubation resulted in decreased trisphosphorylation and accumulation of bis-, mono-, and unphosphorylated forms; the addition of EDTA, which terminates kinase activity via chelation of Mg, stopped dephosphorylation of the kinase (data not shown). These observations are consistent with dephosphorylation of the kinase catalyzed by the kinase itself, which was also observed for the 48-kDa kinase(36) . The present study also indicates that the three core tyrosines are auto-dephosphorylation sites as well as autophosphorylation sites.

Order of Autophosphorylation at the Three Core Tyrosines

Previous studies with the native receptor suggest that phosphorylation of Tyr-1158 is an early event in the autophosphorylation cascade because the predominant species of bis-phosphopeptides is phosphorylated at Tyr-1158 and either Tyr-1162 or Tyr-1163(5, 6, 28) . Tyr-1163 phosphorylation in a Tyr-1162 mutant (replaced by Phe) seems to favor Lys-1165 cleavage versus Arg-1164(35) . As Lys-1165 cleavage was found more efficient for trisphosphorylated kinase than for bisphosphorylated kinase in the case of the wild-type kinase, Tyr-1163 phosphorylation probably occurs late in the cascade. It is not known whether initial phosphorylation occurs at Tyr-1158 or Tyr-1162 or whether it can occur at either site.

To examine the initiation of autophosphorylation at the three core tyrosines of the 35-kDa kinase, the monophosphopeptides (C0 and C1) generated at 2 min of autophosphorylation were recovered from the thin-layer plate and digested with V8 protease (Fig. 7). The predominant product of V8 digestion of the major monophosphopeptide C1 (>90%) corresponds to the peptide TDYYR, which is phosphorylated at Tyr-1162 or Tyr-1163 (>80%), whereas the peptide DIYE, which is phosphorylated at Tyr-1158, represents less than 20%. Similar results were obtained for C0, the minor species of monophosphopeptides (<10%); the peptide TDYYRK, which is phosphorylated at Tyr-1162 or Tyr-1163, is the predominant product of V8 digestion (>90%), indicating that autophosphorylation initiates at Tyr-1162 or Tyr-1163.


Figure 7: V8 protease analysis of mono- and bis-phosphopeptides. Phosphopeptides were isolated from two-dimensional tryptic phosphopeptide maps (Fig. 6) of the 35-kDa kinase at 2 (C0 and C1) and 5 (B2 and B3) min of autophosphorylation. The samples were digested with protease V8, which cleaves the tryptic peptides at Glu-1159. The resulting V8 peptides were separated by thin-layer electrophoresis as described (see ``Materials and Methods''). The positions of the origin and the V8 peptides are indicated. The sequences of the V8 peptides with phosphorylated tyrosine () are shown.



Monophosphorylation of the kinase is immediately followed by bisphosphorylation, as the monophosphorylated form never accumulated to high amounts (maximal level is 30% of the total kinase, Fig. 5B). The bis-phosphopeptides (B2 and B3) generated at 5 min of autophosphorylation were examined by V8 digestion. At this time point, the trisphosphorylated form was undetectable (Fig. 5A). As shown in Fig. 7, V8 digestion of B3 (85% of bis-phosphopeptides) gave two predominant products, one corresponding to Tyr-1158 phosphorylation (42%) and another to Tyr-1162 or Tyr-1163 phosphorylation (55%). The peptide phosphorylated at both Tyr-1162 and Tyr-1163 represents less than 3%. Similar results were obtained for B2 (15% of bis-phosphopeptides). The proportion of the peptides phosphorylated at Tyr-1158, Tyr-1162 or Tyr-1163, or both Tyr-1162 and Tyr-1163 is 44:46:10. Taken together, the bis-phosphopeptides were predominantly phosphorylated at Tyr-1158 and either Tyr-1162 or Tyr-1163, whereas only <5% of the bis-phosphopeptides were phosphorylated at Tyr-1162 and Tyr-1163. Tyr-1158 is thus the major site for the addition of the second phosphate group to the kinase.

Trisphosphorylation of the kinase occurs late in the reaction cascade, as the bisphosphorylated form accumulated to as high as 75% of the total kinase when the trisphosphorylated form appeared at 10 min (Fig. 5B). Tyr-1163 phosphorylation seems to favor cleavage at Lys-1165 versus Arg-1164(35) . To examine the level of Tyr-1163 phosphorylation in the bisphosphorylated kinase that is predominantly phosphorylated at Tyr-1158 and either Tyr-1162 or Tyr-1163, we have compared the ratio of bis-phosphopeptides B2/B3 with that of tris-phosphopeptides A1/A2 (B2 and A1 result from Lys-1165 cleavage, and B3 and A2 are from Arg-1164 cleavage). The ratio of B2/B3 (0.18 ± 0.02) determined at early time points (5 and 10 min) is considerably lower than that of A1/A2 (0.62 ± 0.05) determined at 10, 20, and 30 min, suggesting that the level of Tyr-1163 phosphorylation is relatively lower in the bisphosphorylated kinase compared with the trisphosphorylated kinase. Thus, Tyr-1163 is probably phosphorylated last, after Tyr-1158 and Tyr-1162. Interestingly, the bis-phosphopeptides that were generated at late time points (>90 min) while auto-dephosphorylation of the kinase occurred were found predominantly phosphorylated at Tyr-1162 and Tyr-1163 (70% of bis-phosphopeptides). This is consistent with the finding that the observed ratio of B2/B3 was as high as that of A1/A2 (Fig. 6). Our results thus suggest that the autophosphorylation cascade of the core tyrosines initiates at Tyr-1162, immediately followed by phosphorylation at Tyr-1158 and then at Tyr-1163.

trans-Phosphorylation of the Kinase

Our previous study of the 48-kDa kinase indicated that its autophosphorylation is dependent on enzyme concentration and thus is consistent with the mechanism of trans-phosphorylation(15) . However, evidence for cis-phosphorylation of the cytoplasmic soluble kinase has been reported(37) . Studies with heterotetrameric receptors support trans-(38) , cis-(39) , or both trans- and cis-phosphorylation(40) . To determine the mechanism of autophosphorylation of the 35-kDa kinase, autophosphorylation was carried out over a wide range of enzyme concentrations. Fig. 8shows that the amount of P incorporated into the kinase in 10 min of reaction, determined in the presence of 0.5-9 µM enzyme, 1 mM ATP, and 8 mM MgCl(2) or 4 mM MnCl(2), is dependent on enzyme concentration. Similar results were obtained at 20 or 30 min of incubation or at a lower (0.25 mM) or higher (4 mM) ATP concentration. These results are thus consistent with a trans-phosphorylation mechanism for the 35-kDa kinase. We cannot, however, exclude the possibility that cis-phosphorylation of the kinase could also occur.


Figure 8: Concentration dependence of autophosphorylation of the 35-kDa kinase. Enzyme at indicated concentrations was incubated for 10 min with 1 mM [-P]ATP and 8 mM MgCl(2) (bullet) or 4 mM MnCl(2) (circle). The incorporation of P into the kinase was determined as described in the legend of Fig. 4.



Crystallization of the 35-kDa Kinase

Initial crystals of the 35-kDa kinase (unphosphorylated form, also called apo form) were grown at 21 °C by vapor diffusion in hanging drops containing equal volumes (typically 2 µl) of protein solution (10 mg/ml) and the reservoir solution of 20% PEG 6000 in buffer containing 0.2 M malate/imidazole, pH 7.5. These crystals grew as intergrown plates. Single crystals were obtained by streak seeding freshly prepared drops containing 2 µl of protein solution and 2 µl of 17% PEG 6000 (buffered) and placing them over reservoirs of 13% PEG 6000. Small, single plates were transferred to wash solutions of 16% and then 12% PEG 6000 and afterwards placed (with minimal transfer of solution) in fresh drops containing 2.5 µl of protein solution and 2.5 µl of 16% PEG 6000 over reservoirs of 12% PEG 6000. Macroseeded crystals grew to maximum size over 4-7 days (Fig. 9), typically reaching dimensions 10 times larger than the dimensions of the original seed crystals (50 times 50 times 5 µm). The crystals belong to orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 54.0 Å, b = 73.0 Å, and c = 89.2 Å (when frozen). There is one kinase molecule in the asymmetric unit, and the solvent content, assuming a partial specific volume of 0.73 cm^3 g, is 52%.


Figure 9: Crystal of the 35-kDa kinase obtained by macroseeding. The dimensions are 0.6 times 0.6 times 0.07 mm.



Summary

In conclusion, the 35-kDa kinase, a new soluble derivative of the cytoplasmic protein-tyrosine kinase domain of the insulin receptor, has been engineered on the basis of detailed proteolytic and deletion analysis. This core kinase, produced in large quantities using the baculoviral expression system, exhibits major functional properties of the native receptor kinase domain and is indeed more suitable for biochemical and structural analysis than the previously studied 48-kDa kinase. The presence of only three autophosphorylation sites (three core tyrosines) in the enzyme combined with native PAGE allows one to follow the autophosphorylation cascade of the three tyrosines, thus providing an attractive experimental system for kinetic analysis of autophosphorylation. Finally, the core kinase has been found to be suitable for protein crystallization (Fig. 9), rendering feasible the elucidation of its three-dimensional structure at 2.1-Å resolution by x-ray crystallography(41) . The three-dimensional structure of the core kinase provides new insights concerning the structure and function of the receptor kinase domain, as well as a framework for understanding other members of the protein-tyrosine kinase family.


FOOTNOTES

*
This study was supported in part by grants from the National Institutes of Health (to L. E. and W. A. H.), the Institute of Biosciences and Technology (Texas A & M University) (to L. E.), the W. M. Keck Foundation (to L. E.), the National Science Foundation (to W. A. H.), and the Howard Hughes Medical Institute (to W. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Juvenile Diabetes Foundation International postdoctoral fellowship.

To whom correspondence should be addressed: W. M. Keck Center for Genome Informatics, Inst. of Biosciences and Technology, Texas A & M University, 2121 Holcombe, Houston, TX 77030. Tel.: 713-677-7607; Fax: 713-677-7963.

(^1)
S. R. Hubbard and L. Ellis, unpublished observations.

(^2)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; FYF, peptide RRDIFETDYFRK; FPLC, fast protein liquid chromatography; PEG, polyethylene glycol; bp, base pair(s); kbp, kilobase pair(s).

(^3)
S. R. Hubbard and L. Ellis, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Richard Roth (Stanford University) for the generous gift of the antibody directed against the 48-kDa kinase, Dr. Max Summers (Texas A & M University) for Sf9 cells, and our colleagues in the Ellis lab (especially Dr. Erik Schaefer and Ms. Purita Ramos), Drs. Jeremy Tavaré (Bristol University), Barry A. Levine, Philip Quirk, and Noeleen E. Keane (Birmingham University) for many useful discussions.


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