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. 2013 Jan;9(1):43-50.
doi: 10.1038/nchembio.1118. Epub 2012 Nov 11.

Active site profiling reveals coupling between domains in SRC-family kinases

Affiliations

Active site profiling reveals coupling between domains in SRC-family kinases

Ratika Krishnamurty et al. Nat Chem Biol. 2013 Jan.

Abstract

Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that have a distinct preference for the autoinhibited forms of these kinases. Furthermore, we show that ligands that have this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.

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Figures

Figure 1
Figure 1. An active-site directed probe for ratiometric profiling of protein kinases
(a) The cancer drug dasatinib in complex with the tyrosine kinase SRC (PDB code 3G5D). The arrow shows the site where dasatinib was modified with a benzophenone photo-crosslinker and an orthogonal chemical tag. (b) The chemical structure of probe 1. Probe 1 contains three components: (i) a potent ATP-competitive inhibitor (dasatanib), (ii) a photo-reactive benzophenone crosslinker, and (iii) a hexylchloride tag that selectively labels the active site of the self-labeling protein HaloTag. (c) Experimental crosslinking schematic using 1. Prior to photo-crosslinking experiments, HaloTag is labeled with 1. HT-1 is incubated with a kinase target and then irradiated with UV light. (d). HT-1 efficiently labels the recombinant SRC-family kinases (SFKs) SRC and HCK in cell lysate. Purified SRC or HCK (100 nM) was photo-crosslinked with HT-1 in mammalian cell lysate. Immunoblotting with an anti-SFK antibody shows that a large percentage of SRC and HCK are covalently modified. Upon addition of a dasatinib competitor, no mass-shifted kinases are observed.
Figure 2
Figure 2. Characterization of 1 in cell lysate and live cells
(a) HT-1 labels endogenous SFKs in cell lysates. Immunoblots with an anti-SFK antibody are shown for photo-crosslinking experiments performed with COS-7 and HeLa cell lysates. (b) Endogenous SFKs in live cells transiently expressing HaloTag are photo-crosslinked by 1. COS-7 cells transiently expressing HaloTag were treated with 1 and then irradiated with UV light. Immunoblotting with an anti-SFK antibody shows the presence of mass-shifted SFKs. Addition of an active site competitor prevents photo-crosslinking. (c) 1 labels nonreceptor tyrosine kinases in live cells. V5-tagged nonreceptor tyrosine kinases were coexpressed with HaloTag in COS-7 cells. After photo-crosslinking, the percent crosslinked kinase was determined with an anti-V5 antibody (mean ± SEM, n = 3).
Figure 3
Figure 3. Photo-crosslinking to SFKs that have diverse regulatory domain interactions
(a) The panel of SRC and HCK variants that was generated in this study. Mutations outside the active sites of SRC and HCK were introduced to obtain SFKs with diverse regulatory interactions. Red dots indicate the sites that were modified. (b) The activation loop phosphorylation levels of SFK variants are consistent with their regulatory states. V5-tagged SRC and HCK constructs were transiently expressed in COS-7 cells and the level of activation loop phosphorylation (Tyr416) was determined with an anti-phosphoY416-SFK antibody (phospho-SFK (Tyr416), Cell Signaling). The same samples were immunoblotted with an anti-V5 antibody. (c) 1 labels SFKs with diverse regulatory domain interactions in live cells (mean ± SEM, n = 3). The regulatory states of SFKs have little effect on overall photo-crosslinking efficiency.
Figure 4
Figure 4. Photo-crosslinking competition assays
(a) Chemical structures of the ATP-competitive SFK inhibitors used in photo-crosslinking competition assays with 1. The IC50 values obtained for these inhibitors in photo-crosslinking competition assays with HT-1 and purified SFK constructs are shown in Table 1. (b) Quantitative comparison of the fold differences in cellular competition between activated SFK variants (SRCAct and HCKAct) and their respective SH3eng constructs (SRCSH3eng and HCKSH3eng) for dasatinib and 10.
Figure 5
Figure 5. The catalytic domain of SRC is in the SRC/Cdk-like inactive conformation when bound to 9
(a) Inhibitor 9 (yellow) occupies the ATP-binding site of the catalytic domain of SRC (gray). The pyrazolopyrimidine core sits in the adenine-binding site and makes hydrogen-bonding interactions with the hinge region. (b) The naphthyl-benzyloxy substituent displayed from the C-3 position sits next to the gatekeeper residue (blue) and projects towards helix αC. Helix αC is rotated outwards from the ATP-binding site relative to its position in the active conformation of SRC, which disrupts a salt bridge between the catalytic lysine (Lys295) and Glu310 (both shown in orange). Residues 258–276 have been removed for clarity. (c) Comparison of the relative positions of the helix αCs in the SRC-9 complex (gray) and the active form of SRC (SRC-dasatinib (PDB code 3G5D) (blue)). Helix αC moves approximately 4.5 Å in the SRC-9 complex relative to SRC’s helix αC when bound to dasatinib. The rotation and movement of helix αC displaces a conserved glutamic acid residue (Glu 310; shown in orange) that is important for catalysis, which places SRC in the SRC/Cdk-like inactive conformation. (d) The benzyloxy group of 9 cannot be accommodated in the active form of SRC. Superposition of 9 with SRC in the active conformation (SRC-dasatinib (PDB code 3G5D)) shows a clear steric clash with helix αC.
Figure 6
Figure 6. ATP-competitive SFK inhibitors modulate the SH3 domain accessibilities of SRC and HCK
(a) Representative Western blots for pull-down experiments performed with purified SRCY527F and HCKY527F in the presence of saturating concentrations of various inhibitors. Loaded samples and resin-eluted samples were immunoblotted using an anti-His6 antibody to determine the percentage of loaded kinase that was retained on the PP ligand-containing beads. (b) Quantitation of the pull-down assays performed with SRCY527F and HCKY527F in the presence of each inhibitor that was tested (mean ± SEM, n = 3). The percent of loaded kinase that was eluted from the beads is shown.

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