Studies with chimeric Mpl/JAK2 receptors indicate that both JAK2 and the membrane-proximal domain of Mpl are required for cellular proliferation.
The thrombopoietin (TPO) receptor c-Mpl, like other members of the cytokine receptor superfamily, requires the association and activation of Janus kinases (JAKs) for normal signal transduction. The membrane-proximal portion of the signaling domain, containing conserved box1 and box2 motifs, is sufficient to support the proliferation of cytokine-dependent cell lines and basal megakaryocytopoiesis in vivo. We hypothesized that activation of the JAK2 kinase alone might be sufficient for proliferative signaling. To test this premise, we constructed chimeric receptors in which the extracellular and transmembrane portions of Mpl were fused to the pseudokinase and kinase domains of murine JAK2 kinase. When expressed in the interleukin-3-dependent cell line Ba/F3, the chimeric receptors were appropriately expressed on the cell surface and were able to initiate tyrosine kinase activity upon exposure to TPO. However, chimeric receptors lacking an intact box2 domain of Mpl were unable to support proliferation at any concentration of TPO. Only chimeric receptors containing both JAK2 kinase activity and the box2 region initiated proliferative signaling. Within the box2 motif, we determined that the sequence Glu(56)-Ile(57)-Leu(58) of the Mpl cytoplasmic domain is critical for proliferation of the chimeric receptors. Furthermore, TPO-dependent induction of c-myc transcription is also dependent on this motif. These results indicate that JAK2 activation alone is not sufficient for TPO-induced proliferation and that one or more essential signaling pathways must arise from the cytoplasmic domain of Mpl that includes box2. Although the nature of the signal transduction pathway is not yet known, this second proliferative event is likely to regulate c-myc expression.
Drachman
Jonathan G
JG
Puget Sound Blood Center, Seattle, Washington 98104, USA. jonathand@psbc.org
Miyakawa
Yoshitaka
Y
Luthi
Jennifer N
JN
Dahlen
Debra D
DD
Raney
Alexa
A
Geddis
Amy E
AE
Kaushansky
Kenneth
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002455:Cell Division; D002460:Cell Line; D004789:Enzyme Activation; D016259:Genes, myc; D053614:Janus Kinase 2; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000502:physiology; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D013926:Thrombopoietin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D014443:Tyrosine; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2