Pati Soumya, Gurudutta Gangenahalli U, Kalra Om P, Mukhopadhyay Asok
Stem Cell Biology Laboratory, National Institute of Immunology, New Delhi, India.
BMC Res Notes. 2010 Jan 22;3:14. doi: 10.1186/1756-0500-3-14.
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.
干细胞因子（SCF）受体c-Kit被认为是一种关键的信号分子，它转导干细胞增殖、分化和存活的信号。SCF与其受体的结合触发反式激活，导致激酶和磷酸酶募集到c-Kit催化结构域的对接平台。酪氨酸磷酸酶-1（Shp-1）使“Kit”激酶活性失活/减弱。然而，Kit激活环中的Asp816Val突变以不依赖配体的方式将激酶结构域转变为组成型激活状态（从关闭状态到开启状态）。这种现象完全消除了Shp-1的负调控。为了预测c-Kit与Shp-1之间相互作用的可能分子基础，我们对活化的c-Kit（磷酸化的c-Kit）晶体结构与Shp-1的紧密结构对应物Shp-2的全长晶体结构进行了计算机辅助蛋白质-蛋白质对接研究。
研究揭示了Kit激活结构域中一段保守氨基酸（从Lys818到Ser821），它与磷酸酶的N-sh2和磷酸酪氨酸结合口袋残基形成决定性的氢键。这些氢键可能对c-Kit的催化结构域施加抑制性空间位阻，从而阻止激活环分子与进入的激酶进一步相互作用。我们还预测了Shp-1的SH2结构域中的一个磷酸酪氨酸结合口袋，发现它主要更靠近c-Kit激酶结构域中的类似催化凹槽的结构。
本研究预测，Shp-1的N-sh2结构域与Kit激活环之间的关键氢键可调节Shp-1对c-Kit激酶的负调控。因此，这一发现有望在设计合适的功能获得性c-Kit突变体以诱导造血干细胞条件性增殖方面发挥重要作用。
