A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity.
The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.
Ludwig Institute for Cancer Research, Box 595, Uppsala S-751 24, Sweden.
Federica
F
Chiara
Marie-José
MJ
Goumans
Henrik
H
Forsberg
Aive
A
Ahgrén
Andrea
A
Rasola
Pontus
P
Aspenström
Christer
C
Wernstedt
Carina
C
Hellberg
Carl-Henrik
CH
Heldin
Rainer
R
Heuchel
D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D000077214:Becaplermin; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D015854:Up-Regulation; D014945:Wound Healing
D000199:Actins; D001216:Asparagine; D001224:Aspartic Acid; D000077214:Becaplermin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D010982:Platelet-Derived Growth Factor; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta; D014443:Tyrosine