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. 2006 May 23;103(21):8078-83.
doi: 10.1073/pnas.0601192103. Epub 2006 May 11.

Activation of FIP1L1-PDGFRalpha requires disruption of the juxtamembrane domain of PDGFRalpha and is FIP1L1-independent

Elizabeth H Stover  1 , Jing Chen, Cedric Folens, Benjamin H Lee, Nicole Mentens, Peter Marynen, Ifor R Williams, D Gary Gilliland, Jan Cools
Affiliations

Activation of FIP1L1-PDGFRalpha requires disruption of the juxtamembrane domain of PDGFRalpha and is FIP1L1-independent

Elizabeth H Stover et al. Proc Natl Acad Sci U S A. .

Abstract

Genetic abnormalities that result in expression of chimeric tyrosine kinase proteins such as BCR-ABL1 and ETV6-PDGFRbeta are common causes of hematopoietic malignancies. The paradigm for constitutive activation of these fusion tyrosine kinases is enforced homodimerization by self-association domains present in the fusion partner proteins. The unique interstitial deletion on chromosome 4q12 that leads to expression of the FIP1L1-PDGFRalpha fusion tyrosine kinase was recently identified as a cause of chronic eosinophilic leukemia. In this report, we demonstrate that FIP1L1 is completely dispensable for PDGFRalpha activation in vitro and in vivo. Instead, truncation of PDGFRalpha between two conserved tryptophan residues in the juxtamembrane (JM) domain is required for kinase activation and transforming potential of FIP1L1-PDGFRalpha. The presence of a complete JM domain in FIP1L1-PDGFRalpha is inhibitory, but this autoinhibition can be overcome by enforced homodimerization. Similar effects of the JM domain in the context of PDGFRbeta were observed. These results suggest that disruption of the autoinhibitory JM domain is an alternative, dimerization-independent mechanism by which chimeric tyrosine kinases are constitutively activated and induce leukemogenesis.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Schematic representation of the proteins studied in this work. JM, JM domain; TM, transmembrane domain. “m” indicates the myc epitope tag; “H” indicates the hemagglutinin epitope tag.
Fig. 2.
Fig. 2.
FIP1L1 is not required for the activation of FIP1L1-PDGFRα in vitro. (A) Transformation assay of Ba/F3 cells in the absence of IL3. (B) Phosphorylation of the chimeric PDGFRα proteins when expressed in Ba/F3 cells. (C) Phosphorylation of the chimeric PDGFRα proteins when expressed in 293T cells and general phosphorylation of proteins in 293T cells expressing the different constructs.
Fig. 3.
Fig. 3.
FIP1L1 is not required for the transforming potential of FIP1L1-PDGFRα in vivo. (A) Kaplan–Meier plot showing the short latency of disease onset in FIP1L1-W-PDGFRA and Myc-W-PDGFRA bone marrow transplant assays. (B) Histology of FIP1L1-W-PDGFRA and Myc-W-PDGFRA bone marrow transplant assays showing smears of peripheral blood (magnification: Top, ×100; Inset, ×600; Wright–Giemsa staining) and histopathology in representative sections of spleen and bone marrow (magnification: ×600; hematoxylin/eosin staining). Complete effacement of normal splenic architecture and replacement of normal maturing trilineage hematopoietic elements by a prominent population of maturing myeloid forms, many with folded or ring-like nuclei, is observed.
Fig. 4.
Fig. 4.
Interruption of the JM domain of PDGFRα is required for activation of FIP1L1-PDGFRα. (A) Phosphorylation of the chimeric PDGFRα proteins when expressed in 293T cells and general phosphorylation of proteins in 293T cells expressing the different constructs. (B) Transformation assay of Ba/F3 cells in the absence of IL3 over a period of 4 days. Note that after longer selection cells expressing Myc-WW-PDGFRA also started to proliferate. (C) Phosphorylation of the chimeric PDGFRα proteins when expressed in Ba/F3. Cells were harvested after selection for IL3-independent growth.
Fig. 5.
Fig. 5.
Myc-WW-PDGFRα is an inactive kinase. (A) Structure of the N-terminal part of the Myc-WW-PDGFRα protein, with indications of the positions of the Myc tag, JM domain, and alternative start codon. (B) Transformation assay of Ba/F3 cells in the absence of IL3, showing that Ba/F3 cells expressing the Myc-WW-PDGFRA construct become IL3-independent, whereas the constructs with a mutated internal ATG do not. (C) Phosphorylation of the chimeric PDGFRα proteins when expressed in Ba/F3 cells, showing the absence of phosphorylation of the proteins when the internal ATG is mutated.
Fig. 6.
Fig. 6.
Enforced dimerization can overcome the inhibition by the JM domain. (A) Transformation assay of Ba/F3 cells in the absence of IL3. (B) Phosphorylation of the chimeric PDGFRα proteins.
See this image and copyright information in PMC

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