The structural insights of stem cell factor receptor (c-Kit) interaction with tyrosine phosphatase-2 (Shp-2): An in silico analysis
© Mukhopadhyay et al; licensee BioMed Central Ltd. 2010
Received: 12 August 2009
Accepted: 22 January 2010
Published: 22 January 2010
Stem cell factor (SCF) receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1) deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state), in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit) and full length crystal structure of Shp-2, a close structural counterpart of Shp-1.
Study revealed a stretch of conserved amino acids (Lys818 to Ser821) in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain.
This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.
The c-Kit controls major signaling cascades in hematopoietic stem cells. Earlier study indicated that the catalytic domain of c-Kit consists of many important loop regions, such as catalytic loop (786-796), activation loop (810-839) and substrate-binding loop (829-837), which play crucial role in the activation of Kit kinase domain . Transformation of c-Kit kinase domain from an inactive to hyperactive state is reported to occur on the basis of three distinct molecular phenomena. These are (i) binding of ATP to glycine rich loop residues (596-601), (ii) release of self-inhibitory interaction between substrate binding loop and activation loop, and (iii) loss of intramolecular H-bonds between Lys818-Asp816 and Asn819-Asp816 .
Binding of stem cell factor (SCF) to its receptors triggers dimerization coupled transphosphorylation of c-Kit catalytic domain [2–4], which then emerges with a dual phosphorylated tyrosine containing conserved motif (Y568V569Y570) in human c-Kit . Interestingly, this motif acts as a common docking site for SH2 and SH3 domain containing proteins . The adaptor protein APS, Src family of kinases and Shp-2 tyrosine phosphatase bind to Y568. Similarly, Shp-1 tyrosine phosphatase and the adaptor protein Shc bind to Y570; whereas, C-terminal Src homologous kinase (Chk) and the adaptor Shc bind to both Y568 and Y570 . Shp-1 is known as a negative regulator of various receptors in hematopoietic cells, including c-Kit kinase [8, 9]. Shp-1 and Shp-2 share both sequence and structural homologies . Previous studies have shown that a gain of function mutation, Asp816 to Val816 (a molecular representative of mastocytosis) in Kit activation loop leads to extensive degradation of Shp-1, indicating disruption of Shp-1 and c-Kit interaction [11, 12].
Though SH2 domains of both Shp-1 and Shp-2 are known to interact with c-Kit, the molecular basis of this interaction has not been elucidated. The present study suggests some important residues in Kit activation loop and N-sh2 domain that play crucial role in negative regulation of c-Kit kinase.
Multiple sequence alignment of c-Kit kinase related protein
Multiple sequence alignment of the ligand: Shp-2 and Shp-1 with SH2 domain containing proteins
Docking of Shp-2 and c-Kit cytoplasmic domain
Residues found to be predominantly closer between N-sh2 and c-Kit
Repulsion values for docking (A°)
Residues found closer in Shp-2 bound Kit c-Kit: N-sh2
Distance observed c-Kit: N-sh2 (A°)
Kit Juxtamembrane and PY pocket
Kit Juxtamembrane and N-sh2
Kit activation loop and N-sh2
Kit Juxtamembrane and N-sh2
Kit Juxtamembrane and N-sh2
Kit activation loop and N-sh2
Checking of errors in docked complexes
Examining the substrate-bound complexes of Shp-2 is extremely essential while determining the distances between residues of interacting domains. The analysis showed negligible inter-atomic clashes, zero bumping into each other, and very few intramolecular constraints in Tyr100 of N-sh2. To verify hydrogen bonding, we changed the torsion angle from -156.66 to -116.66 between amino group of Tyr100 and the carboxyl group of Asn819, which showed that the hydrogen bonding of 4.76A° converted to 3.67A° (Additional file 3B).
Analysis of protein-protein interaction
The most striking feature observed in Shp-2 bound Kit complex was the presence of a catalytic cleft like structure made of important residues, like Lys786 to Cys 788, Lys818, Asn819, His 790, Asp792, Thr847, Phe 848, Leu 912, Thr916, Lys 918 and Tyr646, which are highly conserved among c-Kit related kinases (Fig. 3B). These residues were found to be in very close proximity to Tyr100, Ala75, Gly76, Glu79, Met82, Gly83 and His85 of N-sh2, therefore expected to interact to form a stable conformation.
Evaluation of hydrogen bonding between catalytic cleft and SH2 domains
Hydrogen bonds detected between Kit kinase & SH2 domains
Positions of residues in substrate (Donor: c-Kit)
Position of residues in Ligand (Acceptor: Shp-1)
Distance of H-bonds in A°
Our study suggest that subsequent loss of the above intramolecular hydrogen bonding, Lys818 and Asn819 of Kit activation loop might be interacting with conserved PY pocket residue (Ser28 and conserved Tyr100 of N-sh2 domain) for physical blocking of activation loop motifs for further interaction.
The activity of c-Kit is controlled through two important mechanisms: either by juxtamembrane segment auto-inhibition or by phosphatases mediated inhibition. The c-Kit is auto-inhibited by its juxtamembrane segment as it forms a V-shaped loop, inserted directly into the kinase lobes and displaces αC helix of Kit . Subsequent to the activation of Kit kinase, the intramolecular H-bonds between Lys818-Asp816 and Asn819-Asp816 are disrupted, which further destabilizes the activation loop, releasing the steric hindrances associated with the rearrangement of the lobes. This phenomenon allows correct positioning of residues in and from active site, thus opening up the activated structure of Kit kinase (Additional file 4).
In summary, our study has revealed that certain activation loop residues in c-Kit are crucial for interaction of kinase with Shp-1, which may play a vital role in Shp-1 mediated down-regulation of c-Kit kinase activity. We propose that point mutation in Lys818 to Ser821 and Thr847 to Glu849 residues in Kit catalytic domain may lead to the generation of hyperactive functional variants of c-Kit. These may be implicated as efficient gene therapeutic candidates for conditional over-expression in the regeneration of hematopoietic compartments.
Multiple alignments of c-Kit
Using protein information resource (PIR) based multiple sequence alignment tool: PIR-ALN, a phylogenetic tree and multiple sequence alignment were generated for c-Kit and its kinase family members .
Motifs scan of c-kit cytoplasmic domain
Motif Scan search identified short protein sequence in kinase domain of c-kit motifs that are either recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with phospholipid ligands to initiate c-kit mediated cell specific signaling .
Detection of phosphotyrosine binding motifs in SH2 domains of tyrosine phosphatases
The SH2 domain containing proteins were aligned using CLUSTALW for detecting recognition and binding motif in Shp-2, which initiate recruitment of SH2 domains to Kit kinase .
Docking study on c-Kit and Shp-2 interactions and visualization
We have used GRAMM (Global Range Molecular Matching) program to generate Shp-2 bound Kit kinase complexes and predicted H-bond interactions between them. The principle and the methodology of GRAMM are available at the web address http://vakser.bioinformatics.ku.edu/resources/gramm/gramm1/install/readme.pdf. Different repulsion values were applied during docking to determine the best fit complexes (Fig. 3). These repulsion values in A° were determined by three factors, rij (distance between ligand and receptor), U (energy of repulsion) and R, "range" of the potential (the grid step) using the mathematical relationship [29, 30], as shown in the Additional file 5. Distances lesser than 10A°, between important residues (within the predicted interactive zones), decided the best positioning of two proteins for further analysis (Fig. 3). We have used crystal structure of Shp-2 (protein tyrosine phosphatase), 2shp, a full length structural relative of Shp-1, solved at a resolution of 2A°)  and activated crystal structure of human c-Kit cytoplasmic domain (PDB: 1pkg; solved at a resolution of 2.9A° as ligand and substrate) . Analysis of models for the number of inter/intra-molecular clashes, formation of hydrogen bonding and visualization of model images were done by VMD programme.
List of abbreviations
- HCK :
Hematopoietic cell kinase
- LCK :
Lymphocyte Specific Protein Tyrosine Kinase p56
- PY :
Phosphotyrosine binding pocket
- GRB-2 :
Growth factor receptor bound protein 2
- PSSM :
Position-specific scoring matrix
- GRAMM :
Global Range Molecular Matching
- SCF :
Stem cell factor
- Src :
- c-ZAP :
zeta-chain [TCR] associated protein kinase 70 kDa
- JMD :
- APS :
adapter protein with Pleckstrin homology and Src homology two domain.
We are grateful to Prof. R.S. Sowdhamini and Dr. Saikat Chakrabarty of NCBS, Bangalore for extending support in performing the protein-protein interaction studies. Dr. Geetanjali Yadav of NCPGR, New Delhi extended help in analyzing complexes, for that first author is thankful. The first author is indebted to the Director, NII for kind permission to conduct the study.
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