| SHP-1 phosphatase is a member of the Src-homology 2 (SH2) domain containing tyrosine phosphatase (SHP) family. SHP-1 is mainly expressed in hematopoietic cells, where it negatively modulates cytokine-induced signal transduction by deactivation of the Janus kinases (JAK), the main mediators of cytokine signaling. Loss of SHP-1 function has been associated with a number of hematological, myeloproliferative, and autoimmune disorders. Although SHP-1 is implicated in the regulation of many critical cellular functions, the basis for SHP-1 substrate recognition is not well understood. Here, I present a structure-guided study to investigate how SHP-1 recognizes it substrates. Specifically, I aim to characterize the physiologically relevant interaction between SHP-1 and JAK1.;JAK1, one of four JAK family members, has been previously identified as an SHP-1 target in the regulation of interferon (IFN)-α and interleukin (IL)-2 induced signaling. In Chapter 2, I describe the purification of JAK1 kinase domain and validate that JAK1 is an SHP-1 substrate in vitro . In Chapter 3, I describe the characterization of the direct interaction between SHP-1 and JAK1. In this study, I determined and analyzed the co-crystal structure of the catalytic domain of SHP-1 and a JAK1 activation loop peptide in the bis-phosphorylated state to 1.8 Å resolution. A previous study had proposed that SHP-1, similar to tyrosine phosphatase PTP1B, is able to simultaneously bind tandem phosphorylated substrates via a second phosphotyrosine binding site within the catalytic domain. The findings from my study, however, indicate that the catalytic domain of SHP-1 contains a single phosphotyrosine binding pocket, yet prefers the bis-phosphorylated state of the JAK1 activation loop as a substrate. These findings have also suggested a potential mechanism for JAK1 recognition by SHP-1 and have resulted in the identification of a novel structural determinant for substrate recognition among protein tyrosine phosphatases.;In Chapter 4, I present a structural study of the conformational changes that occur upon substrate binding within the catalytic domain of SHP-1, with particular focus on Tryptophan-Proline-Aspartic acid (WPD) loop motif. The WPD loop is a flexible motif that undergoes conformational changes upon substrate binding, thus allowing optimal orientation of the conserved aspartic acid residue for substrate dephosphorylation. In order to structurally characterize the two distinct conformational states of the WPD loop in the SHP family of phosphatases, I determined and analyzed the crystal structures of SHP-1 in the apo state as well as in complex with a phosphate ion, which mimics substrate binding, to resolutions of 1.4 Å and 1.8 Å, respectively. This study led to the first crystal structure of an SHP family phosphatase with the WPD loop in the catalytically active conformation. In addition, I found that the WPD loop in SHP-1 adopts the canonical open and closed conformations observed in other phosphatases in the apo and substrate bound states, respectively. The results from these studies provide new insight into the conformational changes that occur within the catalytic domain of SHP-1 during catalysis and may aid in the development of SHP-1 targeted compounds, which are currently under evaluation for anti-cancer therapy.;Overall, the results presented in my thesis offer new insights into SHP-1 substrate recognition and may aid in the development of SHP-1 targeted drugs. |
