A means to an end: ubiquitination of Mpl

Thrombopoietin (Tpo), via interaction with its cognate receptor Mpl, is the primary cytokine regulating megakaryocyte development and platelet production in addition to its role in hematopoietic stem/progenitor cell (HSPC) homeostasis. Mpl belongs to the type I cytokine receptor family, which includes the erythropoietin receptor (EpoR) and prolactin receptor (PrlR). Ligand binding activates the JAK2 tyrosine kinase, which in turn triggers a cascade of positive and negative signaling events to ensure proper cellular responses. Failure to terminate receptor signaling can lead to oncogenic transformation. Indeed, mutations that constitutively activate Mpl and JAK2 have been found in patients with myeloproliferative neoplasms.¹  Therefore, it is of fundamental importance to understand the mechanisms responsible for restricting cytokine receptor signaling.

One such mechanism that offers swift termination of cytokine signaling involves ubiquitination and internalization of the receptor itself. The mature form of Mpl is subject to rapid internalization, degradation, and clearance from the cell membrane upon Tpo stimulation through both lysosome-dependent and proteasome-dependent mechanisms. In this issue of Blood, Saur et al provide new insights into the turnover of Mpl.²  Specifically, the authors show for the first time that Mpl is ubiquitinated upon Tpo stimulation to regulate Mpl stability, surface expression, and Tpo-mediated cell proliferation in BaF3 cells. The authors identify 2 cytoplasmic lysine residues (K553 and K573) as the targets for ubiquitination. Arginine substitutions of K553 and K557 not only impaired Mpl ubiquitination and degradation, but also conferred hyperproliferation of cells in response to Tpo. Moreover, ubiquitination is shown to be mediated at least in part by the RING domain-containing E3 ligase, c-Cbl. Thus, siRNA-mediated knockdown of c-Cbl, or overexpression of an inactive form of c-Cbl (C379A) mutant, significantly reduced Mpl ubiquitination and turnover. The authors extended their studies to primary cells and found that megakaryocytes and platelets predominantly express the mature form of Mpl, and the Mpl proteins are more stable than those in cell lines with forced Mpl expression. Challenging but rewarding future studies will address to what extent these results apply to Mpl signaling in HSPCs to regulate their expansion and self-renewal in vivo.

Mpl-related receptors such as EpoR and PrlR are also regulated through lysosomal- and proteasomal-mediated internalization and degradation.^3-5  One common mechanism is that the ubiquitinated receptor (often mono-ubiquitinated) is recognized by ubiquitin-binding domain-containing adaptor proteins, which enable receptor internalization, postinternalization sorting, and subsequent degradation. Another mechanism exemplified by interferon receptor 1 (IFNAR1) suggests that ubiquitination at specific lysines increases receptor endocytosis by triggering exposure of an “endocytic motif.”⁶  In all of the above cases, ligand-induced activation of JAK2 and subsequent posttranslational modification of the receptor is required to dock E3 ligases to the receptor to initiate receptor ubiquitination and the down-regulation process.

Surprisingly, the present report found that inhibition of JAK2 fails to impede Mpl ubiquitination or degradation. This contrasts with the authors' previous findings that JAK2 kinase activity is important for Mpl internalization through tyrosine phosphorylation of the Mpl cytoplasmic domain.⁷  It will therefore be of great interest to investigate the JAK2-independent signaling pathway that ubiquitinates Mpl. When expressed in BaF3 cells, the Mpl K_{553 + 573}R mutant that fails to be ubiquitinated showed decreased cell-surface turnover in the absence of Tpo. Whether ubiquitination of Mpl also affects its internalization upon Tpo stimulation requires further elucidation.

c-Cbl docks directly or indirectly through adaptor proteins to phosphorylated receptors upon ligand-induced activation. The fact that JAK2 kinase activity appears dispensable for Mpl ubiquitination raises the question as to how c-Cbl is recruited to the Mpl receptor. Furthermore, when compared with the effects of the Mpl K_{553 + 573}R mutant, loss of c-Cbl had relatively modest effects in promoting Tpo-induced cell proliferation. Together, these results point toward the existence of additional E3 ligases that might account for this difference.

Nonetheless, accumulating evidence supports a role for c-Cbl in constraining signal transduction by multiple receptors. c-Cbl^−/− mice show a marked expansion in HSC numbers with enhanced repopulating activities.⁸  Moreover, c-Cbl^−/− HSPCs exhibit accelerated proliferation and are hypersensitive to Tpo in activating Stat5.⁸  The work from the present report supports the notion that these observations might reflect elevated and/or sustained Mpl surface expression in Cbl^−/− HSPCs. In agreement, c-Cbl mutations found in human myeloid leukemias might exert their concogenic functions by interfering with receptor turnover, thus recapitulating the importance of proper termination of receptor signaling through E3 ligases.^9,10 

National Institutes of Health