Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity

L.A. Cole1,2, R. Zirngibl1,2, A.W.B. Craig1, Z. Jia2 and P. Greer1,2,3,4

1 Cancer Research Laboratories, Departments of 2 Biochemistry and 3 Pathology, Queen's University, Kingston, Ontario, K7L-3N6, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis. Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI. However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity. In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria. Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI. The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains. However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed. The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.

Keywords: Fer/Fps/Fes/kinase/oncogene


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fer protein-tyrosine kinase is closely related to the product of the fps/fes proto-oncogene, and these two cytoplasmic kinases comprise the only known members of protein-tyrosine kinase group IV. Potential roles for Fps/Fes in cytokine receptor signaling (Hanazono et al., 1993aGo,bGo; Izuhara et al., 1994Go; Rao and Mufson, 1995Go; Linnekin et al., 1995Go; Matsuda et al., 1995Go), and for Fer in growth factor or immunoglobin receptor signaling have been proposed (Kim and Wong, 1995Go; Penhallow et al., 1995Go). However, there is little information regarding the molecular basis for the regulation of these kinases, or their association with receptors or potential downstream signaling partners. In addition to their C-terminal catalytic domains, each kinase contains a Src-homology 2 (SH2) domain, and an extended N-terminal region with two putative coiled-coil domains (Kim and Wong, 1995Go; Read et al., 1997Go). Coiled-coils may play an important role in regulating these kinases by promoting interactions with other proteins or by mediating oligomerization. Interestingly, in mice, a presumptive internal promoter directs expression of a testis-specific FerT isoform which lacks this amino terminal domain (Fischman et al., 1990Go). In Drosophila melanogaster a similar fer/ferT gene organization has been described (Paulson et al., 1997Go), while the Caenorhabditis elegans genome appears to encode only the presumptive FerT homologue (Wilson et al., 1994Go).

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., 1996Go; Sicheri et al., 1997Go; Xu et al., 1997Go). 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., 1988Go). 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., 1994Go). However, substitution of this aspartate residue with alanine in cAMP-dependent protein kinase had no affect on activity (Gibbs and Zoller, 1991Go). 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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 Abstract
 Introduction
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 Results
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 References
 
Plasmid constructions

Fer proteins were produced in Cos-1 cells using pXN1, which is a modified version of pECE (Ellis et al., 1986Go) 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 XhoI–XbaI 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 XhoI–NotI fragment in pESNmFerStop was replaced with the XhoI–NotI 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 EcoRI–XhoI 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 XhoI–NotI fragment in this plasmid with the XhoI–NotI fragment from pKSmFerXh/Xb, giving pESXNIImFerK592R.

Sequences encoding a red shifted S65T GFP mutant (Heim et al., 1995Go) were generated by PCR mutagenesis using the Tu #65 GFP cDNA from D.Prasher as a template (Prasher et al., 1992Go). 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 XhoI–NotI 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, 1994Go; Rupp et al., 1994Go) which encodes six copies of the epitope recognized by the 1-9E10 hybridoma (Evan et al., 1985Go). 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. DNA–calcium 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 24–48 h before harvesting for immune complex kinase assays.

Immune complex kinase assays

Cells were washed twice with ice cold TBS-V [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 100 µm vanadate] and harvested by scraping into KLB [20 mM Tris–HCl (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., 1985Go). 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 Tris–HCl (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 5–10 µCi [{gamma}-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 SDS–PAGE 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 Tris–HCl (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., 1996Go). Myc-tagged Fer proteins were detected with 1/20 dilutions of 1-9E10 hybridoma culture supernates. After washing with TBS-T [10 mM Tris–HCl (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 Tris–HCl (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) glutathione–agarose (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 GST–Fer 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., 1997Go). A number of other crystal structure templates, including the human Src tyrosine kinase (Xu et al., 1997Go), 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., 1991Go) and energy minimization was carried out using X-PLOR (Brünger, 1992Go). Diagrams were generated using SETOR (Evans, 1993aGo,bGo).


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 Abstract
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 Materials and methods
 Results
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 References
 
The Fer protein-tyrosine kinase consists of an N-terminal domain, a central SH2 domain, and a C-terminal kinase domain (Figure 1Go). The kinase domain is highly homologous to other protein kinases and shares with them the overall amino acid similarities and subdomain structure, including invariant residues essential for ATP binding such as lysine-592 located in subdomain II, and the presumptive catalytic base aspartate-685 located within subdomain VI. We wished to introduce a kinase-inactivating point mutation into the endogenous murine fer locus in embryonic stem cells as a first step in the production of mice with compromised Fer kinase function. For reasons relating to the exon structure of murine fer and our gene targeting strategy, we choose to introduce an inactivating point mutation into the sequences encoding kinase subdomain IX, rather than the more commonly targeted residues equivalent to lysine-592 or aspartate-685. Kinase alignments showed that aspartate-743 corresponds to a nearly invariant residue among the protein kinase superfamily, suggesting that alteration of this residue might disrupt activity. A crystal structure of the insulin receptor had previously shown that the side chain of the analogous aspartate residue formed two hydrogen bonds with peptide backbone amides at residues in the catalytic loop corresponding to histidine-683 and arginine-684 in Fer (Hubbard et al., 1994Go). This suggested that these interactions might be important for kinase activity by providing conformational stability to the catalytic loop. We reasoned that conversion of aspartate-743 to the larger and oppositely charged arginine residue might destabilize the catalytic loop and disrupt kinase activity. In order to test this idea, we converted the aspartate-743 codon to an arginine codon. This was done in the context of the complete murine fer cDNA using PCR-directed mutagenesis. We also generated a separate Fer mutant in which lysine-592 was converted to an arginine residue. This conservative lysine to arginine substitution had previously been shown to disrupt the activity of other protein kinases, including Fps/Fes. Recombinant Fer proteins were expressed transiently in Cos-1 cells, and tested for activity by immune-complex kinase assays (Figure 2aGo, upper panel). Wild-type Fer displayed robust activity, while both the FerK592R and FerD743R mutants appeared to be catalytically inactive, both in terms of autophosphorylation and phosphorylation of an exogenous substrate (data not shown). However, on longer exposures, we were able to detect some Fer kinase activity in Cos-1 cells transfected with the two Fer mutants, or control cells which had not been transfected. Although this likely represents activity from the low levels of endogenous Fer which could be seen in anti-Fer immunoprecipitates from control cells (Figure 2aGo, lower panel), it could also represent some residual kinase activity in the over-expressed mutants. In order to address this concern we re-engineered the Fer constructs to contain a C-terminal green fluorescence protein (GFP) tag, which would provide a mobility shift in our gel analysis of kinase activity. Wild-type Fer–GFP displayed strong autophosphorylation activity, while the Fer–GFP mutants appeared to be inactive (Figure 2aGo, upper panel). However, once again, longer exposures still revealed low levels of radioactivity at the position of the mutant Fer–GFP proteins (data not shown). As these immune-complex kinases assays were performed using antibody specific to Fer, it was possible that low levels of endogenous Fer were capable of intermolecular phosphorylation of the mutant Fer–GFP proteins. Indeed, anti-Fer immunoblotting of these immune complexes detected Fer-immunoreactive species in addition to the major band migrating at the expected position of Fer–GFP (Figure 2aGo, lower panel). The most prominent of these smaller species was substantially larger than Fer, and likely represents a proteolytic fragment of Fer–GFP. However, there were also detectable amounts of Fer-immunoreactive peptides migrating at the expected position of native Fer.



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Fig. 1. Domain structure of murine Fer. Fer contains two predicted coiled-coil domains (CC) in the N-terminal half, a central Src-homology 2 domain (SH2), and a C-terminal catalytic domain (kinase). Conserved peptide motifs within protein kinase subdomains I through XI are indicated, including those which make up the catalytic loop and activation loop. Residues of interest to this study are indicated (K592, H683 and D743).

 


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Fig. 2. Fer in vitro kinase activity. (A) The indicated Fer or Fer–GFP proteins were immunoprecipitated with anti-Fer antibodies from lysates of transfected cells or untransfected control cells, and subjected to in vitro kinase assays. After resolution on SDS–PAGE and transfer to Immobilon P membrane, Fer proteins were detected by immunoblotting with anti-Fer (lower). Kinase activity was determined by autoradiographic analysis of stripped transfer membranes (upper). (B) The same analysis as described above was carried out on transfected cells expressing the indicated Myc-tagged Fer protein, except anti-Myc antibody was used for immunoprecipitation. (C) The indicated bacterially expressed GST–Fer proteins were isolated on glutathione–agarose and subjected to in vitro kinase assays essentially as described above. GST–Fer protein levels were determined by immunoblotting with anti-Fer antibody (lower) and kinase activity was assessed by autoradiography (upper).

 
In an attempt to avoid immunoprecipitation of endogenous native Fer, we engineered expression constructs to encode Fer proteins tagged with multiple copies of a Myc-epitope at their C-termini. Fer-Myc proteins were then selectively immunoprecipitated from transfected Cos-1 cells using the 1-9E10 anti-Myc monoclonal antibody (Evan et al., 1985Go). These anti-Myc immune-complex kinase assays provided additional evidence that the Fer mutants were catalytically inactive (Figure 2bGo). However, endogenous Fer protein is still likely to be co-immunoprecipitated with the Fer-Myc proteins in these experiments by virtue of homotypic interactions. In a subsequent study we have confirmed that Fer forms homotypic oligomeric structures in vivo, and these interactions potentiate intermolecular autophosphorylation (Craig,A., Zirngibl,R. and Greer,P., manuscript submitted).

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 2cGo). Although comparable levels of wild-type and mutant Fer proteins were isolated on glutathione agarose beads (Figure 2cGo, lower), intrinsic kinase activity was only seen with the wild-type GST–Fer protein (Figure 2cGo, 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., 1997Go), Hck (Sicheri et al., 1997Go) and the FGF receptor (Mohammadi et al., 1996Go). These three kinases, especially Src and Hck, were selected based upon their relatively close amino acid sequence homology to Fer (Figure 3cGo). Perhaps not surprisingly, the predicted structure (Figure 3aGo) was very similar to that of Src (Xu et al., 1997Go). The SH2 domain exists as a separate lobe consisting of a central ß-sheet array flanked by two {alpha}-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 {alpha}F helix. A close up view of the catalytic loop region is also shown (Figure 3bGo). 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., 1994Go), 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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Fig. 3. Modeled Fer structure. (A) The modeled structure of the Fer SH2 and kinase domains based upon the X-ray crystal structure coordinates of Hck, Src and the FGF receptor. The overlaid backbone structures of the Fer wild-type and FerD743R mutant are shown in blue and yellow, respectively. The extended side chains for key residues H683, R684, D685 and D743 (or R743) are indicated in pink for the wild type, and orange for the D743R mutant. (B) A close-up view of these side chains in the catalytic loop region. (C) An amino acid alignment of the SH2 and kinase domains of Fer, Fps, Hck and Src. Sequences corresponding to the predicted {alpha}-helices or ß-sheets shown in (A) are indicated above the alignment, and the approximate limits of protein kinase subdomains I through XI are indicated below the alignment. Stars below the alignment indicate the positions of key residues in the catalytic loop (H683, R684) and in subdomain IX (D743) which are predicted to participate in stabilizing hydrogen bonds. A filled circle below Fer residue Y715 indicates a predicted site of autophosphorylation in the activation loop.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein kinases have been highly conserved through evolution, and this is well illustrated in sequence alignments of a large number of diverse members of this superfamily (Hanks and Quinn, 1991Go; Smith et al., 1997Go). Even before the first protein kinase crystal structures had been solved, sequence alignments had revealed several highly conserved peptide motifs, which provided strong circumstantial evidence for important functional roles of the most highly conserved residues. These conserved peptide motifs define the 11 protein kinase subdomains, and they include nine residues that are largely or completely invariant (Hanks and Hunter, 1995Go). Crystal structures have now confirmed that these residues play key roles in substrate binding, catalysis and conformational stability. The nearly invariant aspartate within subdomain IX appears to fall within the category of residues which provide conformational stability. This residue is located near the beginning of the large {alpha}F helix, and its side chain carboxyl group interacts with the catalytic loop by forming two hydrogen bonds with backbone amides located at residues corresponding to histidine-683 and arginine-684 in Fer. These interactions are predicted to be important for maintaining the correct conformation of the adjacent presumptive catalytic base, aspartate-685. In spite of these predictions, there are two naturally occurring and one engineered example that suggest stabilizing hydrogen bonds involving this subdomain IX aspartate residue are not essential for kinase activity. In CAPL-B, a member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, a threonine residue appears in the place of this aspartate (Beushausen and Bayley, 1990Go); a human cdc2-related kinase has been identified which has an alanine residue at this position (Meyerson et al., 1992Go); and finally, a mutagenesis study performed on Saccharomyces cerevisiae cAMP-dependent protein kinase showed that an alanine substitution at this position had no affect on catalytic activity (Gibbs and Zoller, 1991Go). Interestingly, each of these examples resulted in substitutions with small non-polar or uncharged polar side chains that our model would predict to fit into the same space occupied by the aspartate side chain. Although these side chains could not provide the same stabilizing hydrogen bonds with the catalytic loop, they are not predicted to cause any disruptive steric repulsion. Structural modeling of Fer predicted that several amino acid side chains could fit into the space occupied by this nearly invariant aspartate residue, including alanine and threonine, as well as asparagine, serine, glycine, valine, leucine and isoleucine. One reported sequence of the mouse Lyn kinase does contain an asparagine residue at this position (Stanley et al., 1991Go), however this may have been a cloning artifact or sequencing error, as an updated submission from the same group, as well as two independent submissions from other groups showed an aspartate at this position in murine Lyn (Wilks et al., 1989Go; Yi et al., 1991Go). Although the subdomain IX aspartate is almost completely conserved in the protein kinase superfamily, the hydrogen bonds that it forms with the catalytic loop are not essential for activity. Therefore, as so many other residues could apparently fit into this position without a disruptive affect on the catalytic loop, the high conservation of aspartate at this position is still a mystery. However, our modeling study clearly demonstrated that the large positively charged side chain of arginine could not fit in the available space, and empirical biochemical data showed that the FerD743R mutant, expressed either in mammalian cells or in bacteria, had no detectable kinase activity. Severe steric conflict between the side chains of arginine-743 and histidine-683 was predicted to cause a large localized distortion of the catalytic loop, especially at the position of the histidine and arginine residues immediately preceding the presumptive catalytic base, D685. However, the orientation of D685 did not appear to change, and its position was only slightly shifted. While a small shift in the catalytic base position could have an affect on activity, it is also possible that the large shift and reorientation of H683 and R684 could be responsible for the observed loss of activity. In support of this argument, the residue equivalent to R684 in cAMP-dependant kinase forms hydrogen bonds with phosphate oxygens in the phosphothreonine residue in the activation loop (Knighton et al., 1991Go). These hydrogen bonds are believed to stabilize the active site and permit proper orientation of the peptide substrate.

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., 1997Go; Sicheri et al., 1997Go; Xu et al., 1997Go). 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 {alpha}A helix and K660 in the catalytic domain {alpha}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., 1989Go). The two major phosphorylation sites in human Fps/Fes have been mapped to tyrosines in the activation loop and the {alpha}I helix (Rogers et al., 1996Go). 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 {alpha}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.


    Acknowledgments
 
This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada with funds from the Canadian Cancer Society. P.G. and A.C. are supported by scholarship and postdoctoral fellowship awards, respectively, from the Medical Research Council of Canada. We are grateful to Robert Leggett and Karen Williams for technical assistance.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 7, 1998; revised September 17, 1998; accepted October 14, 1998.