Specific Dephosphorylation Of Janus Kinase Family's Autophosphorylation Sites By Protein Tyrosine Phosphatase

Reversible protein phosphorylation is a homeostasis process.Protein phosphorylation is a post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group from ATP or GTP molecules.The reverse reaction of phosphorylation is called dephosphorylation,and is catalyzed by protein phosphatases.It regulates almost all life activities,such as cell growth and proliferation,differentiation and development,apoptosis,metabolism,protein synthesis,muscle contraction,neural activities and tumorigenesis.The phosphorylation of tyrosine residues,as a more advanced form of evolution and a characteristic of complex multicellular life,is particularly important and prominent in protein phosphorylation.Once the balance of tyrosine phosphorylation in cells is destroyed,many human diseases will occur.So it is very important to strictly control the activities of PTK(protein tyrosine kinase)and PTP(protein tyrosine phosphatase).Janus Kinase(JAK)is an important family of nonreceptor tyrosine kinases and plays an important role in transducing cytokine-mediated signals via the JAK-STAT pathway.The four JAK family members are JAK1,JAK2,JAK3 and TYK2.JAK has an "activation loop" composed of tandem tyrosine residues.The loop plays an important role in activating autophosphorylation and regulating kinase activities.The phosphorylation in tandem autophosphorylation sites is the beginning of JAK activation and the downstream transducing of cytokine-mediated signals via the JAK-STAT pathway thus starts.However,mutations of JAK family kinases,especially excessive activation,are associated with many human diseases.Forexample,the mutation of JAK1 is associated with the development of acute promyelocytic leukemia(APL).The mutation of JAK2 is associated with tumorigenesis in red blood cells and B cells,such as myeloproliferative neoplasms(MPN).The mutation of JAK3 is associated with acute myeloid leukemia(AML),acute lymphoblastic leukemia(ALL)and lymphoma.Since many JAK-based inhibitors have been designed for the common clinical treatment of various immune and tumor diseases,it is particularly important to study the negative regulation mechanism of JAK activities.PTP family members play the most important role in the negative regulation of JAK activities.PTP not only can regulate the phosphorylation of protein kinase products,but also can directly regulate the activity of PTK as the activities of many kinases are regulated by phosphorylation.If these tyrosine residues are dephosphorylated by PTP,the corresponding protein kinases will be inactivated.For example,studies have shown that TYK2 and JAK2 were the true substrates of PTP1 B and demonstrated the role of PTP1 B in the negative regulation of cytokine signaling.As the tandem autophosphorylation sites in the JAK "activation loop" is the beginning of kinase activation and the phosphorylation degree of two tandem autophosphorylation sites will affect the activity of JAK,it is of great significance to reveal the negative regulation mechanism of PTPs to JAK activities by investigating the effect of PTPs on the phosphorylation of JAK tandem autophosphorylation sites.Traditional protein phosphorylation analysis methods include the use of antibodies,radioisotopic labeling,ELISA,fluorescence labeling,etc.However,these traditional methods cannot effectively distinguish two tandem phosphorylation sites.In recent years,mass spectrometry(MS)has been widely used and hasplayed an important role in the analysis of protein phosphorylation for its low noise,high stability and high sensitivity.This paper intended to dephosphorylate short peptides with JAK autophosphorylation sites in vitro by using expressed and purified PTPs.Based on the matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),the dephosphorylation of JAK family tandem autophosphorylation sites by PTPs was analyzed.Research on the dephosphorylation of JAK1,JAK2,JAK3,TYK2 tandem autophosphorylation sites by PTP1 B,TCPTP,SHP-1,SHP-2,He PTP as well as on the prioritizing selection of autophosphorylation sites by different PTPs could provide a theoretical basis for revealing the negative regulation mechanism of different PTPs to JAK family tandem autophosphorylation sites.