1 Cancer Research Laboratories, Departments of 2 Biochemistry and 3 Pathology, Queen's University, Kingston, Ontario, K7L-3N6, Canada
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Abstract |
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Keywords: Fer/Fps/Fes/kinase/oncogene
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Introduction |
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Our understanding of protein kinase structures, functions and mechanisms of regulation has progressed relatively slowly in comparison with the rapid identification of novel members of this protein superfamily. However, structural determinations of a handful of key protein kinases have revealed important insights into how these enzymes perform their catalytic functions, how substrates interact with their active sites, and in some cases how intramolecular interactions contribute to their regulation (Johnson et al., 1996; Sicheri et al., 1997
; Xu et al., 1997
). However, even before any structural information was available, alignments of primary amino acid sequences had already suggested the functional importance of several highly conserved residues within what are known as protein kinase subdomains I through XI (Hanks et al., 1988
). Among these, an aspartate residue in kinase subdomain IX is nearly invariant within the protein kinase superfamily. Structural studies suggested that hydrogen bonds between the side chain carboxyl group of this aspartate and two backbone amides in the catalytic loop, provide conformational stability to the active site (Hubbard et al., 1994
). However, substitution of this aspartate residue with alanine in cAMP-dependent protein kinase had no affect on activity (Gibbs and Zoller, 1991
). As alanine could not participate in the same hydrogen bonding that aspartate does, this result demonstrated that these stabilizing hydrogen bonds are not essential for kinase activity. We show here that conversion of the equivalent aspartate in the Fer protein-tyrosine kinase to an arginine residue completely abolished enzymatic activity. A modeling analysis of Fer provided a structural basis for this dramatic affect on kinase activity, and also explained why substitution of this aspartate with alanine or other amino acids would be unlikely to affect kinase activity. Thus, a role in conformational stabilization of the active site would not appear to justify the high degree of conservation seen at this subdomain IX aspartate residue in the protein kinase superfamily.
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Materials and methods |
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Fer proteins were produced in Cos-1 cells using pXN1, which is a modified version of pECE (Ellis et al., 1986) in which the XbaI cloning site was converted to a NotI site by digestion with XbaI, and ligation with an annealed NotI adapter oligo (XNot: 5'-CTA GGC GGC CGC-3').
The 2.7 kb murine fer cDNA from plasmid pF2c.8 was excised with ApaI and DraI, blunt-ended with Klenow and subcloned into the SmaI site of pECE. The resulting pECEmFer plasmid was kindly provided by K.Letwin and T.Pawson. Fer coding sequence from nucleotide 2079 through to the 3'-UTR was excised from pECEmFer as an XhoIXbaI fragment, and cloned into the corresponding sites in pKS+, to give pKSmFerXh/Xb. This plasmid was used to recover the natural Fer termination codon and 3'-UTR sequences in some of the plasmid constructions described below.
Wild-type mFer was PCR amplified from pECEmFer using Pfu thermostable DNA polymerase (Stratagene) and oligo #879 (mFer5'UTRSal: 5'-GCT GTC GAC CAC AGT GTG GAG GAT AAG-3') and oligo #880 (mFerStopNot: 5'-GCC CTG GGC GGC CGC TGT GAT CAT CTT C-3'). After digestion with SalI and NotI, this PCR product was cloned into pXN1 between the SalI and NotI sites, giving the plasmid pESNFerStop. The NotI site at the 3' end of the fer sequence replaces the Fer termination codon and facilitates in-frame insertion of C-terminal coding sequences.
To express wild-type Fer, the XhoINotI fragment in pESNmFerStop was replaced with the XhoINotI fragment from pKSmFerXh/Xb, giving pESXNmFer. The replacement fragment spans Fer coding sequence from nucleotide 2079, through the natural termination codon and into the 3'-UTR sequences.
To express the FerD743R mutant, PCR mutagenesis was first used to generate the D743R mutation using oligo #881 (mFerXhofor: 5'-GTT GTA TCT CGA GAG CAA GAA CTG C-3') and mutagenic oligo #1460 (5'-CCA CAC TCG CGA TTC AGA ACT GTA TCT CC-3') to amplify a 5' fragment from nucleotide 2072 through the point of mutation, and mutagenic oligo #1459 (5'-TCT GAA TCG CGA GTG TGG AGC TTC GGC ATC C-3') and the NotI-containing oligo #927 (mFer3'TAG: 5'-GGA GCC GCG GCC GCA CTA TGT GAT CAT C-3') to amplify a 3' fragment spanning from the point of mutation to a novel NotI site immediately after the termination codon. Aliquots of the two PCR reaction were then mixed, and the external oligos #881 and #927 were used to amplify the complete fragment. This was digested with XhoI and NotI, and cloned between the corresponding sites in pESXNmFer, giving pESXmFerD743R.
To express the FerK592R protein, PCR mutagenesis was first used to generate the K592R mutation using oligo #872 (Fer-987: 5'-GCG GAA GAG CTC ACA CAG ACC CAG-3') with mutagenic oligo #874 (Fer-1838: 5'-CTT GCA CGT TCT AAT GGC AAC AGG-3') to amplify sequences from nucleotide 987 through the point of mutation, and mutagenic oligo #873 (Fer-1815: 5'-CCT GTT GCC ATT AGA ACG TGC AAG-3') with oligo #875 (Fer 2507: 5'-GAC AGT GAG CTC TTT GTG AAG GTC-3') to amplify sequences from the point of mutation to nucleotide 2507. Aliquots of these PCR reactions were mixed and amplified by PCR using the exterior oligos #872 and #875. The resulting PCR product was digested with SacI, and the fragment spanning nucleotides 996 through 2498 was cloned into pECEmFer between the SacI sites at 996 and in the 3' multiple cloning site. From this intermediate plasmid, an EcoRIXhoI fragment spanning nucleotides 495 to 2084 was used to replace the corresponding sequences in pESNmFerStop, giving pESNmFerK592R. The normal termination codon and 3'-UTR was recovered by replacing the XhoINotI fragment in this plasmid with the XhoINotI fragment from pKSmFerXh/Xb, giving pESXNIImFerK592R.
Sequences encoding a red shifted S65T GFP mutant (Heim et al., 1995) were generated by PCR mutagenesis using the Tu #65 GFP cDNA from D.Prasher as a template (Prasher et al., 1992
). The 5' end was amplified with a NotI-containing oligo #706 (5'-AAG GCG GCC GCG ATG AGT AAA GGA GAA-3') and an antisense mutagenic oligo #765 (5'-C ACC ATA AGT GAA AGT AGT GAC AA-3'), while the 3' end was amplified using a sense mutagenic oligo #764 (5'-ACT ACT TTC ACT TAT GGT GTT CAA-3') and a NotI-containing oligo #707 (5'-AT TGC GGC CGC GGA CAT TTA TTT GTA-3'). Aliquots of these two PCR reactions were combined and amplified in a PCR reaction using the two external oligos #706 and #707. After digestion with NotI, this S65T mutant GFP-encoding sequence was cloned into the NotI site in pXN1 to give the GFP expression plasmid pXN-GFP.
To generate the FerGFP expression plasmid, the GFP encoding NotI fragment from pXN-GFP was cloned into the NotI site at the end of the Fer coding sequence in pESNFerStop to produce the plasmid pESNmFerGFP.
To generate the FerK592RGFP expression plasmid, PCR mutagenesis was used to produce the FerK592R mutation using oligo #872 (Fer-987: 5'-GCG GAA GAG CTC ACA CAG ACC CAG-3') with mutagenic oligo #874 (Fer-1838: 5'-CTT GCA CGT TCT AAT GGC AAC AGG-3') to amplify sequences from nucleotide 987 through the point of mutation, and mutagenic oligo #873 (Fer-1815: 5'-CCT GTT GCC ATT AGA ACG TGC AAG-3') with oligo #875 (Fer 2507: 5'-GAC AGT GAG CTC TTT GTG AAG GTC-3') to amplify sequences from the point of mutation to nucleotide 2507. Aliquots of these PCR reactions were mixed and amplified by PCR using the exterior oligos #872 and #875. This PCR product was digested with KpnI and XhoI and the fragment spanning nucleotide 1434 to 2084 was then used to replace the corresponding sequences in pESNmFerGFP, giving pESNmFerK592RGFP.
To generate the FerD743RGFP expression plasmid, the sequences encoding the FerD743R mutation were amplified from the pGEXmFerD743RHis plasmid (described below) using oligo #881 (mFerXhofor: 5'-GTT GTA TCT CGA GAG CAA GAA CTG C-3') and oligo #880 (mFerStopNot: 5'-GCC CTG GGC GGC CGC TGT GAT CAT CTT C-3'). The XhoINotI fragment of this PCR product was then used to replace the corresponding sequences in pESNmFerGFP, giving pESNmFerD743RGFP.
Expression plasmids encoding Myc epitope-tagged Fer proteins were generated by replacing the GFP-encoding sequences in the Fer-GFP expression plasmids with a sequence derived from the pCS2+MT plasmid (Turner and Weintraub, 1994; Rupp et al., 1994
) which encodes six copies of the epitope recognized by the 1-9E10 hybridoma (Evan et al., 1985
). EagI site-containing oligos Myc5' (5'-CTT GCG GCC GTT GCA GGA TCC CAT CG-3') and Myc3' (5'-CT AGC GGC CGC GAC TCA CTA TAG TTC TAG-3') were used to amplify a 320 base pair sequence from pCS2+MT. EagI-digested PCR product was used to replace the GFP-encoding NotI fragments of pESNmFerGFP, pESNmFerK592RGFP and pESNmFerD743RGFP, giving pESNmFerMyc, pESNmFerK592RMyc and pESNmFerD743RMyc, respectively.
The pGEX-2T plasmid (Pharmacia LKB Biotechnology) was modified by digestion with EcoRI and ligation with the annealed oligos EcoH6+ (5'-AAT TCA TCA CCA TCA CCA TCA CTA-3') and EcoHis (5'-AAT TTA GTG ATG GTG ATG GTG ATG-3'). In the resulting plasmid, pGEX-His, the multiple cloning site is restored and six histidine codons followed by a termination codon are added downstream of the EcoRI site. The pGEX-His plasmid was used to produce GST-fusion proteins which also contain C-terminal oligohistidine tracts. Murine fer cDNA sequences encoding the SH2 and kinase domains were amplified using oligos #1492 (mFerSH2: 5'-GGT GGA TCC CCC CTG GCG GAG CAT-3') and #1462 (mFer3'His6: 5'-GGC GAA TTC CTG TGA TCA TCT TCT TGA TG-3'). After digestion with BamHI and EcoRI, this PCR product was cloned into pGEX-His between the BamHI and EcoRI sites. The resulting pGEXmFerwtHis plasmid encodes a fusion protein consisting of N-terminal GST, murine Fer residues P455 to T823 and a C-terminal oligohistidine tract. PCR mutagenesis was used to make pGEXmFerD743RHis. Mutagenic oligo #1460 (5'-CCA CAC TCG CGA TTC AGA ACT GTA TCT CC-3') and oligo #1492 were used to PCR the 5' end of the sequence, and mutagenic oligo #1459 (5'-TCT GAA TCG CGA GTG TGG AGC TTC GGC ATC C-3') and oligo #1462 were used to PCR the 3' end of the sequence. Aliquots of these two PCR reactions, and the two exterior oligos (#1492 and #1462) were then used to amplify the complete sequence encoding the mFerD743R sequence. This PCR product was then digested with BamHI and EcoRI, and cloned into pGEX-His as described above. The pGEXmFerK592RHis plasmid was generated using the same approach as described for the wild type, except the cDNA template used in the PCR was the pESNmFerK592R mutant described above. The mutants were checked by sequencing.
Transfections
Cos-1 cells were seeded onto multi-well 35x10 mm tissue culture dishes (Falcon) at a density of 120 000 cells per 35mm well, giving approximately 25% confluence for transfections the following day. DNAcalcium phosphate precipitates were prepared in 12x75 mm polypropylene tubes (VWR) by mixing 2 µg of plasmid DNA in 200 µl of 0.25 M CaCl2 with 200 µl 2xBBS solution [50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.06]. DNA precipitates were allowed to form for 60 min at room temperature, then resuspended and added dropwise to cells in 35 mm wells containing 2.5 ml of DMEM supplemented with 10% FBS. After overnight incubation the media was replaced and cells were cultured for an additional 2448 h before harvesting for immune complex kinase assays.
Immune complex kinase assays
Cells were washed twice with ice cold TBS-V [10 mM TrisHCl (pH 7.5), 150 mM NaCl, 100 µm vanadate] and harvested by scraping into KLB [20 mM TrisHCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P40, 0.5% (v/v) sodium deoxycholic acid, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µM sodium orthovanadate, 100 µM phenylmethylsulfonylfluoride]. Cell lysates were clarified by centrifugation at 14 000 g for 20 min at 4°C. Soluble lysates were then added to 25 µl of 50% (v/v) protein G conjugated to Sepharose CL-4B which had been preincubated with 200 µl of 1-9E10 anti-myc hybridoma culture supernate (Evan et al., 1985). After mixing on a nutator platform for 2 h at 4°C, immune complexes were collected by brief centrifugation, and washed 5 times with KLB, followed by one wash with KRB [20 mM TrisHCl (pH 7.5), 10 mM MnCl2, 100 µM sodium orthovanadate]. Kinase reactions were performed by resuspending the washed immune complex with 30 µl of KRB supplemented with 510 µCi [
-32P]ATP and incubating for 20 min at 30°C. The reactions were terminated by addition of 30 µl 2xSDS sample buffer and heating at 100°C for 5 min. Proteins were then resolved on SDSPAGE gels, and transferred to Immobilon-P membrane (Millipore) using a semidry apparatus (BioRad). Membranes were blocked overnight at 4°C with Blotto [5% Carnation skim milk powder in 10 mM TrisHCl (pH 7.5), 150 mM NaCl]. Fer proteins were detected by incubation at room temperature for 2 h with 1/500 dilutions of rabbit polyclonal anti-Fer-LA antibody raised against a GST fusion protein containing murine Fer amino acids L97 to A382 (Haigh et al., 1996
). Myc-tagged Fer proteins were detected with 1/20 dilutions of 1-9E10 hybridoma culture supernates. After washing with TBS-T [10 mM TrisHCl (pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween-20], membranes were incubated with a 1/10 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (for anti-Fer) or goat anti-mouse IgG (for anti-myc) secondary antibodies (Vector Laboratories) in TBS-T for 1 h at room temperature. After washing with TBS-T, immune complexes were detected using enhanced chemiluminescence (NENTM Life Science Products). The membranes were stripped by incubation in 62.5 mM TrisHCl (pH 6.7), 2% (w/v) SDS, 100 mM 2-mercaptoethanol at 65°C for 30 min followed by washing in TBS-T. Kinase activity was determined on stripped membranes by autoradiography.
Isolation and analysis of GST-Fer fusion proteins
Bacteria harboring pGEX-Fer expression plasmids were grown overnight in LB-amp (Luria broth containing 125 µg/ml ampicillin). For inductions of GST-Fer proteins, fresh prewarmed LB-amp was inoculated with a 1/10th volume of overnight culture, grown for 1 h, then supplemented with 100 µM IPTG and grown for another 2 h. Bacteria was harvested by centrifugation at 10 000 g for 10 min at 4°C. Soluble GST-Fer proteins were prepared by sonication of bacterial pellets in ice cold KLB, followed by centrifugation at 14 000 g for 20 min at 4°C. GST-Fer proteins were affinity purified by addition of 1% (v/v) glutathioneagarose (Sigma Chemical Company), followed by mixing on a nutator platform for 30 min at 4°C and washing five times with KLB. In vitro kinase activity and detection of GSTFer fusion proteins was preformed as described above for immune complex kinase assays.
Structural modeling
The primary template for our modeling was the crystal structure of the Hck tyrosine kinase determined at 2.6 Å resolution (Sicheri et al., 1997). A number of other crystal structure templates, including the human Src tyrosine kinase (Xu et al., 1997
), were also considered in the modeling. The sequence of the model was changed to the corresponding Fer sequence. Insertions and deletions were made where necessary. After native Fer was modeled, various mutations and the subsequent modeling of the displaced catalytic loop were carried out. The resulting models were then energy minimized. The graphic and modeling program used was FRODO (Jones et al., 1991
) and energy minimization was carried out using X-PLOR (Brünger, 1992
). Diagrams were generated using SETOR (Evans, 1993a
,b
).
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Results |
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In order to conclusively assess the intrinsic kinase activity of these wild-type or mutant Fer proteins without the complication of endogenous Fer activity, we expressed them as glutathione-S-transferase (GST)-fusions in bacteria, where there is no endogenous expression of Fer, or any other tyrosine kinase. Fusions consisted of N-terminal GST fused to the SH2 and kinase domains of Fer, but they lacked the natural N-terminal Fer domain. These proteins were isolated from bacterial lysates by affinity chromatography on glutathione agarose beads, and subjected to in vitro kinase assays followed by immunoblotting with anti-Fer antibody (Figure 2c). Although comparable levels of wild-type and mutant Fer proteins were isolated on glutathione agarose beads (Figure 2c
, lower), intrinsic kinase activity was only seen with the wild-type GSTFer protein (Figure 2c
, upper).
In order to better understand the effect of the D743R substitution on Fer kinase activity, we modeled the structure of murine Fer based on the known coordinates of Src (Xu et al., 1997), Hck (Sicheri et al., 1997
) and the FGF receptor (Mohammadi et al., 1996
). These three kinases, especially Src and Hck, were selected based upon their relatively close amino acid sequence homology to Fer (Figure 3c
). Perhaps not surprisingly, the predicted structure (Figure 3a
) was very similar to that of Src (Xu et al., 1997
). The SH2 domain exists as a separate lobe consisting of a central ß-sheet array flanked by two
-helices. A long linker region connects the SH2 domain to the catalytic domain. The active site, which includes the catalytic loop, is located between the two lobes of the catalytic domain. The predicted structure shows the superimposed conformations of both the wild type and D743R mutant, with the extended side chains of residues H683, R684 and D685 in the catalytic loop, and the D743 or R743 residues near the beginning of the
F helix. A close up view of the catalytic loop region is also shown (Figure 3b
). In the wild-type structure, the peptide backbone, colored blue, is deep within the cleft of the active site, while the peptide backbone of the D743R mutant, colored yellow, is shifted considerably away from the original position. The side chains in the wild-type structure (shown in pink) illustrate how D743 points up toward the catalytic loop. As described in the insulin receptor (Hubbard et al., 1994
), two hydrogen bonds form between the D743 side chain carboxyl group and backbone amides at H683 and R684. However, in the D743R mutant, the arginine side group (shown in orange) extends much further toward the catalytic loop, where it would have to occupy the same position as the H683 side chain. The resulting van der Waal's repulsions were predicted to cause a substantial shift of the peptide backbone in the D743R mutant. The D743R mutant model also predicted a dramatic reorientation of the H683 and R684 side chains (shown in orange). Interestingly, the position and side chain orientation of D685, the presumptive catalytic base, is only slightly shifted. When we modeled the structures of other amino acids substituted into the D743 position, it was apparent that several residues including alanine and threonine would be permitted in this position without disrupting the conformation of the catalytic loop.
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Discussion |
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Unfortunately, the Fer model provides little information regarding intramolecular interactions involving the SH2 domain. Unlike the Src and Hck structures, the SH2 domain of Fer cannot interact with a C-terminal tail tyrosine phosphorylation site, as no such site exists in the corresponding Fer tail region. In Src and Hck, the linker region between the SH2 and catalytic domain interacts with both the SH3 and catalytic domains. These interactions are predicted to play an important role in the regulation of Src-family kinases (Moarefi et al., 1997; Sicheri et al., 1997
; Xu et al., 1997
). In contrast, Fer does not have an SH3 domain, and our modeled Fer structure does not predict any interactions between the linker region and the catalytic domain. We did see an electrostatic interaction between E473 in the SH2
A helix and K660 in the catalytic domain
E helix; however, this interaction may not actually occur in Fer as the linker region is likely to allow more substantial flexibility than that seen in Src and Hck. Increased flexibility around the linker region might allow for other more stable intramolecular interactions not seen in this model, including interactions between the SH2 domain and tyrosine phosphorylation sites. In this regard it is interesting to note that partial proteolysis analysis of the related Fps/Fes kinase has provided evidence for intramolecular interactions involving the SH2 domain (Koch et al., 1989
). The two major phosphorylation sites in human Fps/Fes have been mapped to tyrosines in the activation loop and the
I helix (Rogers et al., 1996
). The phosphorylation sites in Fer have not yet been described, and although the activation loop site is a likely candidate, the tyrosine residue in the
I helix of human Fps/Fes is not conserved in Fer.
There is relatively little information regarding the biological function of the Fer kinase. The wide spread expression pattern suggests a function common to many cell types, and the close structural similarity with the Fps/Fes kinase suggests they may have similar, and perhaps redundant biochemical functions. We have recently targeted the murine fps/fes gene with a kinase-inactivating amino acid substitution and found that homozygous mutant mice are viable with only subtle phenotypic defects (Senis,Y., Zirngibl,R., McVeigh,J. and Greer,P., manuscript submitted). We have therefore embarked upon a similar genetic analysis of fer function involving the production of mice expressing a catalytically inactive Fer kinase. Our initial characterization of the murine fer locus suggested that the exon organization is similar to that of the closely related fps/fes gene, but with much larger introns. A further consideration in designing a fer targeting strategy is the existence of a testis-specific ferT promoter, which is probably located somewhere within the fer gene. As a preliminary step in disrupting murine fer, we wished to determine if Fer kinase activity could be compromised by introducing an amino acid substitution mutation into sequences contained in the penultimate fer coding exon. The most promising amino acid residue in these sequences was the aspartate residue in subdomain IX. We have demonstrated that a nonconservative substitution to an arginine residue completely abolished Fer kinase activity. By targeting the murine fer gene with this mutation, we expect to disrupt the normal biological function of both Fer and FerT in a manner that is unlikely to be rescued by other kinases with redundant biochemical functions, including the closely related Fps/Fes kinase. Phenotypes in mutant Fer mice, and compound mutants of both Fps/Fes and Fer, should help clarify the biological functions of these two closely related protein tyrosine kinases.
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Acknowledgments |
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Notes |
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References |
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Received July 7, 1998; revised September 17, 1998; accepted October 14, 1998.