Structural determinants of the gain-of-function phenotype of human leukemia-associated mutant CBL Oncogene

Scott A. Nadeau, Wei An, Bhopal C. Mohapatra, Insha Mushtaq, Timothy A. Bielecki, Haitao Luan, Neha Zutshi, Gulzar Ahmad, Matthew D. Storck, Masashi Sanada, Seishi Ogawa, Vimla Band, Hamid Band

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Abstract

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express secondsite point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr3Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr 3 Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBLY371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-offunction in which mutantCBLproteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyperactivate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

Original languageEnglish (US)
Pages (from-to)3666-3682
Number of pages17
JournalJournal of Biological Chemistry
Volume292
Issue number9
DOIs
StatePublished - Mar 3 2017

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Hematopoietic Stem Cells
Oncogenes
Proline
Protein-Tyrosine Kinases
Leukemia
Stem cells
Phenotype
Stem Cell Factor
Colony-Stimulating Factor Receptors
Phospho-Specific Antibodies
Phosphotyrosine
Phosphorylation
Ligases
Ubiquitin
Carrier Proteins
Mutation
Proteins
Myeloid Leukemia
Fusion reactions
Cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Structural determinants of the gain-of-function phenotype of human leukemia-associated mutant CBL Oncogene. / Nadeau, Scott A.; An, Wei; Mohapatra, Bhopal C.; Mushtaq, Insha; Bielecki, Timothy A.; Luan, Haitao; Zutshi, Neha; Ahmad, Gulzar; Storck, Matthew D.; Sanada, Masashi; Ogawa, Seishi; Band, Vimla; Band, Hamid.

In: Journal of Biological Chemistry, Vol. 292, No. 9, 03.03.2017, p. 3666-3682.

Research output: Contribution to journalArticle

Nadeau, SA, An, W, Mohapatra, BC, Mushtaq, I, Bielecki, TA, Luan, H, Zutshi, N, Ahmad, G, Storck, MD, Sanada, M, Ogawa, S, Band, V & Band, H 2017, 'Structural determinants of the gain-of-function phenotype of human leukemia-associated mutant CBL Oncogene', Journal of Biological Chemistry, vol. 292, no. 9, pp. 3666-3682. https://doi.org/10.1074/jbc.M116.772723
Nadeau, Scott A. ; An, Wei ; Mohapatra, Bhopal C. ; Mushtaq, Insha ; Bielecki, Timothy A. ; Luan, Haitao ; Zutshi, Neha ; Ahmad, Gulzar ; Storck, Matthew D. ; Sanada, Masashi ; Ogawa, Seishi ; Band, Vimla ; Band, Hamid. / Structural determinants of the gain-of-function phenotype of human leukemia-associated mutant CBL Oncogene. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 9. pp. 3666-3682.
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