Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.
It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.
Department of Laboratory Medicine and Pathology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada.
Peng
P
Wang
Fang
F
Wu
Jingdong
J
Zhang
Todd
T
McMullen
Leah C
LC
Young
Robert J
RJ
Ingham
Liang
L
Li
Raymond
R
Lai
D045744:Cell Line, Tumor; D004789:Enzyme Activation; D006801:Humans; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000209:etiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D012694:Serine; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases