Screening And Functional Analysis Of Proteins Interacting With Cytoplasmic Domain Of Mpl

Thrombopoietin (TPO) is a critical hematopoietic cytokine regulating megakaryopoiesis and platelet production. Cooperateing with other cytokines, it can support the differentiation and survival of hematopoietic stem cell (HSC). TPO acts by binding to its cell surface receptor, Mpl. It is important to study the signal transduction of Mpl, which can make us well understand the function of TPO.TPO binding induces receptor homodimerization, leads to activation of the Janus kinase (JAK2) and phosphorylation of the tyrosine residues of Mpl, and then activates multiple downstream signal transduction pathways. Recently, it was reported that a mutated Mpl, of which the proximal-membrane ten amino acid residues in the cytoplasmic domain were deleted, could transduce downstream signal of TPO such as Raf, MAPK, PI3K without the activation of JAK2. Moreover, eliminating all of the tyrosine residues of Mpl doesn't destroy TPO's ability to support celluar proliferation in TPO-dependent cell line. These data suggest that there may have some other mechanisms for TPO signal transduction.Our aim is to indentify new proteins that can physiologically interact with intracellular domain of Mpl. The yeast two-hybrid screen was performed firstly, and sixteen gene sequences were selected from the Pretransformed MATCHMAKER Mouse 12.5-day Embryo cDNA library when using the cytoplasmic domain of mouse Mpl as bait. Fourteen of the sixteen sequences were further confirmed by mammlian two-hybrid assays and the other two sequences were excluded. After function and location analysing, eight gene sequences were selected to study further. The eight full-length genes were amplified by RT-PCR and then subcloned into corresponding two-hybrid vectors. Results showed that these full length proteins could still associate with Mpl in mammlian two-hybrid system, respectively. Among these genes, we have more interests in two: RACK1 and Atp5d.RACK1 is the receptor for activated protein kinase C, serving as a scaffold and linking PKC to its substrates. It has been reported that RACK1 may act as a scaffold or adaptor protein involved in the recruitment, assembly, and/or regulation of a variety of...