Phosphoproteomic Analysis Identifies Deregulated Expression of Acting Signaling Networks in T Cell Lymphomas
Carlos Murga-Zamalloa, Delphine Rolland, Yoon Jeon, Venkatesha Basrur, Kojo Elenitoba-Johnson, Megan Lim. University of Michigan, Ann Arbor, MI
Background: Proteins involved in actin dynamics play a key role in lymphocyte biology and have been implicated in lymphomagenesis. Actin polymerization in T-cells is tightly regulated by phosphorylation of key proteins such as WASP and CRKL. Identification of novel phosphoproteins would be critical for studying the signaling networks involved in lymphocyte biology and lymphomagenesis. In order to identify actin signaling networks which are activated in T-cell lymphomas, we carried out mass spectrometry-based proteomic analyses to identify phosphoproteins in T-cell lymphoma derived cell lines.
Design: Phosphorylated peptides of CTCL (HH, HUT78 and MAC1), ALK+ ALCL (SUPM2, SUDH1L and KARPAS 299) and NK lymphoma (NK92MI and YT) cell lines were enriched using 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). Proteins involved in actin regulation and signaling networks were identified using DAVID and STRING software, respectively. Hierarchical cluster analysis was performed with MeV software.
Results: Serine, tyrosine and threonine phosphorylated peptides from sixty one proteins involved in the regulation of the actin cytoskeleton were identified. Phosphorylation motif analysis indicated that many of these peptides are targets of GSK-3, GRK-1 and Casein kinase 1. Proteins associated with lymphomagenesis such as TWF1, CRKL, WASP and a role in migration (paxillin and zyxin) were identified to be differentially phosphorylated. A subset of proteins was specific to distinct disease categories; GRLF1 in NK lymphoma; CASL in CTCL and VASP in ALCL. Interestingly, hierarchical clustering using the sixty one proteins grouped the ALK+ ALCL cell lines distinct from the others. Protein network analysis revealed that WASP and NCK1 are located at the center of numerous pathways highlighting their potential functional significance. Western blot analysis confirmed the expression of a subset of the proteins.
Conclusions: Our phosphoproteomic and bioinformatics analysis identified numerous phosphoproteins involved in the regulation of actin dynamics to be differentially expressed in distinct subtypes of T cell lymphoma. Importantly, ALK+ ALCL expressed a distinct signature of actin cytoskeletal phosphoproteins that may be important for the biology of these tumors. Our data reveal the utility of unbiased phosphoteome interrogation of T cell lymphomas in characterizing actin associated signaling networks.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 276, Monday Morning