The structural insights of stem cell factor receptor (c-Kit) interaction with tyrosine phosphatase-2 (Shp-2): An in silico analysis
TL;DR: It is predicted 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, which is expected to play a significant role in designing suitable gain-of-function c- Kit mutants for inducing conditional proliferation of hematopoietic stem cells.
Abstract: 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.
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TL;DR: A critical role of Shp2 is revealed in maintenance of a functional HSC/progenitor pool in adult mammals, at least in part through a kinase-phosphatase-kinase cascade.
89 citations
TL;DR: The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c- Kit that makes it independent of SHP-1/SHP-2 negative regulation.
Abstract: Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein–protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation.
10 citations
01 Jan 2012
TL;DR: The present report has introduced a novel outlook for hematopoietic stem cells (HSCs) therapy on the basis of structural and functional interpretation of stem cell factor receptor (c-Kit), which can change the perspectives of oncogene-oriented structural biology approach.
Abstract: During last decade or more, investigations have been pursued towards fi nding molecular cross-talks involving proto-oncogene transformation to oncogene in aggressive form of hematological malignancies. Oncologists and structural biologists have identi fi ed several hotspots in tyrosine kinase proto-oncogenes, which decided the diverse aberrant cell fate decisions in cancers. But direct exploitation of the aberrant behavior of tyrosine kinase oncogenes in regenerative cell therapies has not been thought, so far. The present report has introduced a novel outlook for hematopoietic stem cells (HSCs) therapy on the basis of structural and functional interpretation of stem cell factor receptor (c-Kit), which can change the perspectives of oncogene-oriented structural biology approach. c-Kit, the master signal transducer of HSCs holds many structural intricacies, for which it becomes vulnerable to the extent of oncogenic activation or inactivation. Both these attributes strictly involve in deciding on the fate of HSCs signaling. c-Kit hyperactivity contributes to oncogenic signaling of HSCs, leading to hematological malignancies. Whereas, c-Kit inactivation leads to irreversible developmental defects. Conserved hotspots in c-Kit structure have long been discussed by cell biologists as well as structural biologists, as these sites delineate the sensitivity towards kinase inhibitors or anticancer drugs. Recent studies of our group envisaged a new dimension to c-Kit’s interaction with one of its negative regulators. The study suggested crucial hydrogen bonds between c-Kit catalytic domain and tyrosine phosphatase-1 (SHP-1) regulate its kinase activity. So, disruption of any such bonding between c-Kit and its negative regulator may lead to hyperactive variants. Study also suggested an interesting hypothesis, which explains transient ectopic expression of hyper-activating Kit-mutant can induce early commitment of hematopoiesis in lethally irradiated mice. We have hypothesized that designing non-oncogenic or partial hyper-activating mutants in c-Kit may generate new class of active variants. This approach may lead to the generation of therapeutic candidates for HSCs speci fi c molecular targets in regenerative medicine using other existing proto-oncogenes.
8 citations
TL;DR: The effect of over‐expressing c‐kit on early recovery of the haematopoietic compartment, in irradiated hosts, is investigated.
Abstract: Objectives: Stem cell factor receptor, c-kit, is considered to be the master signalling molecule of haematopoietic stem cells. It develops the orchestral pattern of haematopoietic cell lineages, seen by its varying degree of omnipresence in progenitors, lineage committed and mature cells. We have investigated the effect of over-expressing c-kit on early recovery of the haematopoietic compartment, in irradiated hosts.
Materials and methods: Normal bone marrow cells (BMCs) were transfected with Kitwt (wild-type c-kit) or its variant Kitmu (asp814tyr) by electroporation. Lethally irradiated mice were transplanted with normal or transfected congeneic BMCs. The effect of ectopic expression of c-kit on haematopoietic cell recovery was determined by analysing donor-derived cells. Furthermore, effects of both types of c-kit over-expression on progenitor and lineage-committed cells were examined by flow cytometric analysis of Sca-1 and lineage-committed (Lin+) cells respectively.
Results: Hyper-activating Kitmu significantly improved recovery of the haematopoietic system in irradiated hosts. In vivo results showed that the donor-derived c-kit+ cell population was increased to more than 3-fold in the case of Kitmu-transfected cells compared to normal and Kitwt over-expressing BMCs. In general, survival of progenitor and committed cell was improved in the Kitmu over-expressing system compared to the other two cohorts.
Conclusion: These results suggest that recruitment of the hyper-activating variant of c-kit (Kitmu) lead to early recovery of the bone marrow of lethally irradiated mice.
2 citations
01 Jan 2011
TL;DR: A novel signaling mechanism of kinase- phosphatase-kinase cascade in HSCs/progenitors in adult mammals is revealed and it is shown that Shp2 regulates coordinately multiple signals, including AKT, MAPK and STAT3 pathways, to promote Kit expression via the transcriptional factor Gata2.
Abstract: It is now widely accepted that the fine balance between hematopoietic stem cell (HSC) self-renewal and differentiation is required for blood cell homeostasis and is evidently orchestrated by the dynamic interplay between environmental cues and intrinsic genetic setup. Nevertheless, how the niche signal-initiated intracellular signaling cascades are regulated in HSCs is less understood. The Stem cell factor (SCF)/Kit system has served as a classical model in deciphering molecular signaling events in the hematopoietic compartment. It has long been known that the microenvironment, or the so- called niche, of Sl/Sl mouse (steel-Dickie mice) that harbors a mutation in membrane-bound SCF or Kit-ligand, is not capable of supporting normal HSC functions. Study of various white spotting (W) mutant mice revealed an essential role of SCF-Kit signaling in HSC survival and quiescence maintenance. Kit is now a most frequently used marker for HSCs and progenitors. However, it remains to be elucidated how Kit expression is regulated in HSCs. In this dissertation, we report that a cytoplasmic tyrosine phosphatase Shp2, acting downstream of Kit, promotes Kit gene expression, constituting a Kit-Shp2-Kit signaling loop. Inducible ablation of none-receptor protein tyrosine phosphatase number 11 (PTPN11/Shp2) in adult hematopoietic compartment led to severe cytopenia in bone marrow, spleen and peripheral blood in mice. Shp2 removal resulted in elevated HSC death and loss of HSC quiescence, causing suppression of the functional HSCs/progenitors pool. Shp2- deficient HSCs failed to reconstitute lethally irradiated recipients in a cell autonomous manner due to combined defects in homing, self-renewal and survival. We show that Shp2 regulates coordinately multiple signals, including AKT, MAPK and STAT3 pathways, to promote Kit expression via the transcriptional factor Gata2. Therefore, this study reveals a novel signaling mechanism of kinase- phosphatase-kinase cascade in HSCs/progenitors in adult mammals
References
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TL;DR: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved and modifications are incorporated into a new program, CLUSTAL W, which is freely available.
Abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
63,427 citations
"The structural insights of stem cel..." refers background in this paper
...motif in Shp-2, which initiate recruitment of SH2 domains to Kit kinase [28]....
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TL;DR: Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands, allowing segments of biological pathways to be constructed in silico.
Abstract: Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.
1,619 citations
"The structural insights of stem cel..." refers background in this paper
...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 [27]....
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TL;DR: The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains.
893 citations
"The structural insights of stem cel..." refers background or methods in this paper
...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°) [13] and activated crystal structure of human c-Kit cytoplasmic domain (PDB: 1pkg; solved at a resolution of 2....
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...Amongst SH2 and SH3 domain containing proteins epidermal growth factor receptor, platelet derived growth factor receptor, PLCg, N/C-sh2, Abl kinase, Src family kinases and phosphatases (Shp-1 and Shp-2) are important [13-15]....
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TL;DR: The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction, and comparison with the structure with the high affinity complex reveals only localized and relatively small changes.
748 citations
"The structural insights of stem cel..." refers background in this paper
...It is similar to SH2 domain bound phosphotyrosine peptides complexes, as shown by NMR and X-ray structural studies [21,22]....
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...The functionally important residues in this motif initiate strong decisive interactions between guanidinium group of invariant arginine residue at a conserved position in SH2 and anionic PY phosphate group, as shown in structure of p85 subunit of PI3 kinase [21]....
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TL;DR: A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma.
Abstract: A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000 The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus
629 citations
"The structural insights of stem cel..." refers background in this paper
...The second reason may be due to change of JMD mediated auto-inhibition of kit kinase to its hyper-activated state [18-20]....
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