The Membrane-proximal Region of the Thrombopoietin Receptor Confers Its High Surface Expression by JAK2-dependent and -independent Mechanisms

Tong, Wei; Sulahian, Rita; Gross, Alec W.; Hendon, Natalie; Lodish, Harvey F.; Huang, Lily

doi:10.1074/jbc.m607524200

Cited by 32 publications

(31 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found that the N-terminal SH2-proximal region of this linker, residues P 501 KPKDKSNLLVF, was essential for interaction of JAK2 with the EpoR. These residues may be involved in direct binding to the membrane-proximal Box 1 region of cognate receptors or may hold the JAK N-terminal segment in the correct conformation to interact with cognate receptors (9,36,50). Four gain-of-function mutations in this linker transformed Ba/F3 cells, and three alanine-scanning mutants resulted in increased JAK2 activity.…”

Section: Discussionmentioning

confidence: 93%

“…EpoR surface expression was measured in the presence of wild-type or mutant full-length JAK2 or GST-JAK2(N) constructs in half a million ␥2A cells, as described previously (36).…”

Section: Jak2-dependent Epor Surface Expression-ha-taggedmentioning

confidence: 99%

“…Activation-Four activating mutations lie in the SH2-pseudokinase linker, previously demonstrated to be part of the JAK2 N-terminal segment necessary and sufficient for interaction with cytokine receptors to promote their surface expression (9,36). We hypothesized that this linker may act to coordinate ligand stimulation of the cytokine receptor to JAK2 activation.…”

Section: The Jak2 Sh2-pseudokinase Linker Is Essential For Interactiomentioning

confidence: 99%

See 2 more Smart Citations

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

Zhao

Dong

Zhang

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The Janus family of tyrosine kinases (JAKs) 2 are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogenactivated protein kinase, and phosphatidylinositol 3-kinase/ AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3-5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11-13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14 -18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19,20) and caused erythrocytosis in a murine bone marrow transplant model (14,(21)(22)(23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24 -26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokineinduced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction w...

show abstract

Section: Discussionmentioning

confidence: 93%

“…EpoR surface expression was measured in the presence of wild-type or mutant full-length JAK2 or GST-JAK2(N) constructs in half a million ␥2A cells, as described previously (36).…”

Section: Jak2-dependent Epor Surface Expression-ha-taggedmentioning

confidence: 99%

Section: The Jak2 Sh2-pseudokinase Linker Is Essential For Interactiomentioning

confidence: 99%

See 1 more Smart Citation

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

Zhao

Dong

Zhang

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…12,28,29 To test whether JAK2 is required for TpoR activation by CALR mutants, we performed STAT5 transcriptional assays in the human JAK2-deficient g2A cells in the absence of JAK2 or using a TpoR receptor mutant that does not bind JAK2. CALR mutants could not induce TpoR activation in the absence of JAK2 (Figure 2A).…”

Section: Jak2 Is Required For Tpor Activation By Calr Mutantsmentioning

confidence: 99%

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

Chachoua

Pecquet

El-Khoury

et al. 2016

Blood

278

369

View full text Add to dashboard Cite

show abstract

“…[8][9][10] Prior work has shown that the canonical ER-Golgi route for trafficking of Mpl to the cell surface is aberrant in myeloproliferative neoplasms, linked in part to requirements for WT Jak2 acting as a chaperone. 11,12 An alternative pathway to the surface for Mpl is provided by an unconventional autophagy-linked secretory pathway. 13 It is important to determine the functional impact of CAMT mutations on Mpl signaling and trafficking, because clinical presentation, disease progression, and treatment options reflect the underlying cellular mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

et al. 2017

View full text Add to dashboard Cite

Key Points• We report unique familial cases of CAMT presenting with a novel MPL W272R mutation in the background of the activating MPL K39N mutation.• Function of mutant Mpl receptor can be rescued using 2 approaches: autophagic cell surface delivery and CRISPR-Cas9 gene editing.Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T(K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin.Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34 1 cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

show abstract

The Membrane-proximal Region of the Thrombopoietin Receptor Confers Its High Surface Expression by JAK2-dependent and -independent Mechanisms

Cited by 32 publications

References 34 publications

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

Contact Info

Product

Resources

About