Wiskott-Aldrich Protein Is Phosphorylated by NPM-ALK and Regulates the Proliferation and Invasion of Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) is a subtype of mature T cell lymphoma. The NPM-ALK fusion protein is expressed in 80% of pediatric ALCL and arises from a chromosomal translocation t(2;5)(p23;q35). NPM-ALK is a constitutively active oncogenic tyrosine kinase that regulates the function of numerous proteins involved in apoptosis and cell cycle progression. In order to better understand the mechanisms of NPM-ALK mediated oncogenesis, we utilized an unbiased mass spectrometry –based phosphoproteomic approach to identify novel downstream signaling targets of the oncogenic NPM-ALK.

The phosphoproteome was enriched using a two-step procedure including immobilized metal affinity chromatography (IMAC) and phospho-tyrosine immunoaffinity purification and subsequently analyzed by liquid chromatography and high mass accuracy tandem mass spectrometry (LC-MS/MS). Western blot analysis with phospho-specific antibodies and in vitro kinase assays were used to validate the MS results. Stable lentiviral transduction of shRNA vectors was used to evaluate the functional consequences. Cell proliferation was measured using the WST-1 assay. Effect on transformation capacity was assessed with colony formation assay using methyl-cellulose agar. Invasive properties were assessed using collagen based transwell assays and adhesion was evaluated by attachment onto fibronectin substratum.

Phosphoproteomic analysis identified several proteins involved in actin dynamics to be regulated by ALK tyrosine kinase activity. These include WASP, CRKL, NCK1, ARP2/3 and ITSN2. Pathway network analysis situated WASP at the center of the actin network. Western blot analysis demonstrated that phosphorylation of WASP at Y290 is regulated by NPM-ALK. Expression of wild type NPM-ALK increased the phosphorylation of WASP Y290 in 293T cells compared to a kinase-defective K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates WASP at Y290 as well as a novel residue Y102. The interaction between NPM-ALK and WASP was demonstrated using immunoprecipitation of co-transfected 293T cells as well as in ALCL cell lines. Because Y102 residue of WASP is located in the domain involved in interaction with its chaperone WIP, we evaluated whether its phosphorylation regulates the interaction. The interaction of WASP and WIP was decreased when Y102 was phosphorylated as demonstrated by immunoprecipitation of phospho-mutants of WASP (Y102F and Y102E). Surface plasmon resonance studies with recombinant WT or phosphomimetic (Y102E) WASP and WIP confirmed these observations. Furthermore, cycloheximide chase experiments demonstrated that phosphorylation of WASP at Y102 decreased it protein stability. Stable WASP knockdown in SUDHL1 cells led to reduced adhesion to fibronectin (21% ± SEM 0.4% vs 13% ± SEM 0.6%, p<0.01) and invasion through collagen (68% ± SEM 16% vs 12% ± SEM 3%, p<0.05). Interestingly, stable knock down of WASP resulted in increased proliferation (0.79 RFU ± SEM 0.07 vs 0.54 RFU ± SEM 0.019, p<0.01) and colony formation (58 colonies ± SEM 1.2 vs 26 colonies ± SEM 3.3, p< 0.01) of SUDH1L cells.

Our results show that NPM-ALK regulates the phosphorylation of a network of proteins involved in actin dynamics, one of which is WASP. NPM-ALK directly phosphorylates WASP at residues Y290 and Y102 and thereby regulates WASP function in actin dynamics critical for cell adhesion, invasion and proliferation.

No relevant conflicts of interest to declare.