Homology Alignment and Ala Scanning Identify a Potential Substrate-DockingSite in Csk. Csk contains an SH3 and an SH2 domain (27, 28).Previous studies suggest that the SH3 and SH2 domains do notdirectly contribute to substrate recognition by Csk (29, 30).The Csk catalytic domain is composed of an ATP-binding lobeand a peptide-binding lobe (19, 20). We focused our search forthe substrate-docking site on the peptide-binding lobe, whichcontains 180 residues (271–450). Because Csk and Chk arethe only two PTKs capable of phosphorylating SFKs on Ytail,the docking site must be conserved in Csk and Chk but not inother PTKs. To locate the structural features uniquely conservedin Csk and Chk, primary sequences of Csk (28), Chk (31), Src(32), and Hck (33) in the peptide-binding lobe were aligned.This alignment identified 29 residues that are uniquely conservedin the Csk family (Fig. 1). Because Csk phosphorylation of kdSrcis highly sensitive to ionic inhibition (data not shown), wefirst focused on 14 polar and charged residues (Arg-279, Arg-281,Arg-283, Glu-303, Arg-318, Asp-325, Ser-331, Lys-337, Asp-344,Lys-362, Ser-381, Lys-393, Lys-401, and Glu-438). Among the14, Ser-331 is buried in the interior of Csk, and Arg-318, Lys-337,and Asp-344 have been previously excluded as possible residuesto interact with Src (24, 34, 35), leaving 10 residues as potentiallypart of the substrate-docking site. No obvious spatial patternwas observed when the uniquely conserved residues were mappedonto the tertiary structure of Csk.
To determine which of these residues may be part of a substrate-dockingsite, they were individually mutated to Ala and the mutant enzymeswere purified to apparent or near homogeneity. For some reason,one of the mutants, Arg359Ala, was not produced. By functionalnature of the hypothetical substrate-docking site, we reasonedthat this site would be critical for Csk activity toward itsphysiological substrate but not important for phosphorylationof an artificial substrate. Thus, as the initial screening,the kinase activities of the mutants toward an artificial anda physiological substrate were determined (Fig. 2A). The artificialsubstrate used was polyE4Y, a random co-polymer of Glu and Tyrin the ratio of 4:1. PolyE4Y lacks a defined phosphorylationsite or higher orders of structure and is commonly used as ageneric PTK substrate. The activity toward polyE4Y, therefore,is considered as general kinase activity. The physiologicalsubstrate used is a recombinantly expressed kinase-defectiveSrc (kdSrc) that contains a Lys295Met mutation. The mutationinactivates Src but does not affect its ability to serve asa specific substrate for Csk or Chk (10, 18). The use of kdSrcinstead of active Src eliminates interference to the assay bySrc autophosphorylation.
If a residue is part of the substrate-docking site of Csk, itsmutation to Ala will likely more dramatically decrease Csk activitytoward kdSrc than toward polyE4Y. Three of the 10 mutants, Arg279Ala,Arg281Ala, and Arg283Ala, displayed this property, with thelatter two exhibiting >80% of WT activity toward polyE4Ybut <20% of WT activity toward kdSrc. Lys362Ala displayedthe opposite effect, having a more dramatic effect on polyE4Yphosphorylation than kdSrc phosphorylation. This is representativeof a group of Csk mutants that preferentially affect phosphorylationof polyE4Y over kdSrc, which were separately characterized (unpublisheddata). Mutation of the other residues had similar effects onpolyE4Y and kdSrc phosphorylation. Overall, Ala scanning mutagenesisimplicated Arg-281, Arg-283, and Arg-279 as part of the substrate-dockingsite.
Additional Residues Are Identified to Be Critical for Src Phosphorylation.Arg-279, Arg-281, and Arg-283 are located on -helix D, a shorthelix located near the active site of Csk. In the tertiary structure,these three residues form a triangle (Fig. 2B). Each side ofthis triangle measures 15–16 Å. Two other residues,Ser-280 and Phe-382, although not uniquely conserved in Cskfamily, also fall within or near the area defined by the Argtriangle and could be part of the substrate-docking site. Theywere individually mutated to Ala, and the ability of the mutantsto phosphorylate kdSrc and polyE4Y was determined. Ser280Alaand Phe382Ala displayed significantly less relative activitytoward kdSrc than toward polyE4Y (Fig. 2C), indicating thatthese two residues are also part of the substrate-docking site.Ser-284 and Ser-381 are located just outside of the Arg triangle(Fig. 2B). The mutation of either one to Ala affected polyE4Yand kdSrc phosphorylation equally (Fig. 2C), indicating thatthese two residues were not specifically important for Src recognition.
Two residues, Ser-273 and Asp-276, are located on the -helixD, and between the Arg triangle and the active site (Fig. 2B).If the Arg triangle and the active site of Csk form a continuousbinding surface for Src interaction, then these residues arealso likely to be important for Src phosphorylation by Csk.To test this possibility, they were mutated to Ala and the mutantswere purified and analyzed (Fig. 2C). Ser273Ala displayed WTlevel activity toward polyE4Y and 70% WT activity toward kdSrc.Asp276Ala retained 40% of WT activity toward either polyE4Yor kdSrc. This result suggests that Ser-273 is also part ofthe substrate-docking site.
The above Ala scanning study identified six residues (Ser-273,Arg-279, Ser-280, Arg-281, Arg-283, and Phe-382) as specificallyimportant for kdSrc phosphorylation. The first five are locatedon -helix D, and the last one is located next to the helix inthe tertiary structure. Several residues immediately outsidethis region were not specifically important for kdSrc phosphorylation.Therefore, these six residues represent the major determinantsof the substrate-docking site.
Mutations of the Putative Substrate-Docking Site Convert Cskinto a Generically Active Kinase Unable to Phosphorylate, Regulate,or Bind to Src. To further verify the substrate-docking site,two mutants, one containing double mutations (DM) of Arg281Ala,and Arg283Ala and the other containing quadruple mutations (QM)of Ser280Ala, Arg281Ala, Arg283Ala, and Phe382Ala, were generated.Ser-273 and Arg-279 were not included in these mutants, becausemutation of each had relatively minor effects on kdSrc phosphorylation(Fig. 2). Fig. 3A compares the Lineweaver-Burk plots of WT andmutant enzymes using kdSrc as the variable substrate. Both mutantshad dramatically decreased kcat and mildly higher Km values.Table 1 summarizes the catalytic parameters of the two mutantsusing both kdSrc and polyE4Y as the variable substrates. TowardkdSrc, the Km increased 1.5-fold for DM and 3-fold for QM. Bothmutants had kcat values <15% of that of WT, resulting ina 20-fold or more reduction in kcat/Km ratio. In contrast, themutants had similar kcat and moderately increased Km towardpolyE4Y, resulting in a reduction of kcat/Km ratio by <3-fold.This result demonstrates that the mutated residues are specificallyimportant for kdSrc phosphorylation.
Because the Csk mutants were defective in phosphorylating kdSrc,it was expected that they would be defective in regulating Srcby phosphorylation. This possibility was tested by a Src inactivationassay (14) (Fig. 3B). Src expressed in and purified from insectcells (36, 37) was incubated with WT or mutant Csk (equal polyE4Ykinase activity were used for WT or mutant Csk) in the presenceof ATP and MgCl2 for 10 min, and then the Src activity in theincubation was determined. If Csk or the mutants were able toinactivate Src, the preincubation would result in a decreasein Src activity. As expected, WT Csk inactivated Src, but thetwo mutants did not. This result confirmed that the mutationsconverted Csk into a generically active kinase without the abilityto recognize and phosphorylate Src.
If the mutated residues in DM and QM are indeed the substrate-dockingsite, the mutants should have a much weaker interaction withkdSrc than WT Csk. Pull-down assays were performed, as describedin Methods, to evaluate the interaction of Csk to kdSrc (Fig. 4).GST-wtCsk, but not GST, was able to pull down kdSrc (closeto a 1:1 ratio), indicating that kdSrc was binding specificallyto Csk. Similarly, Csk mutants S273, K393, and K401 were alsoable to pull down kdSrc, correlating to their significant residualkdSrc kinase activity. In contrast, DM, QM, R284, and F382 werenot able to pull down kdSrc, in good agreement with their inactivityto phosphorylate kdSrc. This result further confirmed that theidentified docking residues were specifically responsible forbinding to Src. Because these residues are responsible for bindingto Src and rendering Csk effective in phosphorylating Src, weconclude that these residues constitute the major determinantsof the substrate-docking site.
Peptidic Mimic of the Substrate-Docking Site Specifically Inhibitsthe Phosphorylation of kdSrc by Csk. As another independenttest for the identified substrate-docking site, the abilityof peptides mimicking the docking site as inhibitors for Cskwas determined. Peptides mimicking the substrate-docking sitestructure would be expected to compete against Csk in bindingto Src. Thus, they should inhibit Csk phosphorylation of kdSrcbut not that of polyE4Y. To test this idea, three peptides weresynthesized: VDYLRS (P1), RSRGRS (P2), and RSVLGG (P3). Theycover residues V275 through Ser-280 (P1), Arg-279 through Ser-284(P2), and Arg-283 through Gly-288 (P3). Note that P2 containedfour residues, Arg-279, Ser-280, Arg-281, and Arg-283, thatare key residues of the substrate-docking site of Csk. In contrast,P1 or P3 contained only at most two residues important for SFKbinding. P1 and P3 (up to 1 mM) did not inhibit Csk phosphorylationof polyE4Y or kdSrc (data not shown). In contrast, P2 potentlyinhibited Csk phosphorylation of kdSrc (IC50 = 21 ± 3.8µM) but only moderately inhibited polyE4Y phosphorylation(IC50 = 422 ± 46 µM) (Fig. 5). The differentialinhibition of kdSrc and polyE4Y phosphorylation indicated thatP2 specifically interfered with the interaction between Cskand kdSrc. This result further indicates that the residue clusterlocated on -helix D and mimicked by P2 contained key determinantsof the substrate-docking site.