Proteomic Studies of Anaplastic Large Cell Lymphoma
Megan S. Lim
University of Utah School of Medicine
Salt Lake City, UT
Anaplastic Large Cell Lymphoma
Anaplastic large-cell lymphoma (ALCL) is a distinct subtype of peripheral T-cell lymphomas harboring
chromosomal translocations involving the ALK tyrosine kinase. The t(2;5)(p23;q35) chromosomal aberration
resulting in overexpression of a chimeric oncogene, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK),
is the most common translocation found in these tumors. The resulting NPM-ALK fusion protein has been
well documented as a constitutively active tyrosine kinase and the causative oncogene in t(2;5) positive
ALCLs. The NPM-ALK protein plays a key role in ALCL lymphomagenesis and has been shown to cause lymphoid
malignancy in vitro and in vivo.
The emerging field of proteomics provides a powerful avenue to carry out functional studies of
protein-protein interactions and characterization of signal transduction pathways. This is in large part
due to advances in protein sample preparation, analytical sensitivity of mass spectrometers and
improvements in instrument software and protein databases ) . Until recently, assessment of interacting
proteins has only been feasible via laborious and time-consuming molecular biologic techniques such as
the yeast-two-hybrid system. Using a variety of approaches including protein complex purification,
immunoprecipitation, affinity chromatography followed by high performance liquid chromatography (HPLC)
and electrospray ionization and tandem mass spectrometry (ESI-MS/MS), interacting proteins of CD4
receptor complex and protein kinase Ce signaling complex have been identified.
The recent development of multidimensional liquid chromatographic methods combined with tandem mass
spectrometry (LC-LC-MS/MS) has permitted sensitive detection of low abundance proteins, membrane proteins
and proteins with extreme isoelectric points (pI). The ability to perform global quantitative proteomics
has been significantly enhanced by the advent of the isotope-coded affinity tag-based technology (ICAT™
) which is efficient in simplifying the proteome, and in combination with 3-D LC-MS/MS permits detection
and quantification of proteins and peptides from very complex samples.
Our laboratory has utilized a functional proteomic approach to study anaplastic large cell lymphomas.
Identification of NPM-ALK interacting proteins by tandem mass spectrometry
Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular
signals, and are potential targets for novel therapies. Using a functional proteomic approach, we
determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation
with anti-ALK antibody followed by electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). A
total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal
antibodies while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in
the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS and PLCγ1.
More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with
potential NPM-ALK interacting protein domains were identified. These include adaptor molecules (SOCS,
Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated
kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit) and
heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by western blotting and
reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and
peptide identification by nanoflow ESI-LC/MS/MS for the high-throughput identification of proteins within
the ALK signaling complex and potential definition of its signaling pathways.
Quantitative proteomic analysis of
differentially expressed proteins induced by NPM-ALK overexpression
The global molecular and cellular consequences of NPM-ALK overexpression are largely unknown. In
addition, the identity and function of only a limited number of downstream molecules important for its
oncogenic activity are currently understood. We have used a functional quantitative proteomic approach
to determine the global effects of NPM-ALK expression on cell function. Jurkat cells were transfected
with a plasmid containing the NPM-ALK chimeric gene. The NPM-ALK overexpressing Jurkat cells were
compared to those expressing the vector only. Quantitative analyses of differentially expressed proteins
were determined by isotope-coded affinity tagging (ICAT™) followed by liquid chromatography
(LC) and tandem mass spectrometry (MS/MS). Equivalent quantities of total cell lysates obtained from the
NPM-ALK transfected cells and the vector control cells were ICAT™ labeled, and subjected to
avidin affinity chromatography. Offline fractions were collected, digested with trypsin, and analyzed by
automated reverse phase nanospray LC-MS/MS. Some 124 proteins showed a 1.5 fold or greater change in the
NPM-ALK positive cells as compared to the vector control cells. Of these, 79 proteins were unregulated
by greater than 1.5-fold while 45 proteins were downregulated by greater than 1.5-fold. Differential
expression of selected proteins was validated by western blot analyses. Analysis of functional groups
of proteins demonstrated upregulation of protein kinases, cytoskeletal proteins and proteins associated
with proliferation and translation. In addition, upregulation of proteins previously reported to be
important mediators of the ALK signaling pathway including, PLCγ1, Ki-67, GRB2, Jak2, and PI3-K were
observed. This study, for the first time, reveals the global proteomic consequences of NPM-ALK
overexpression as a singular molecular abnormality and provides novel insight into the signal
transduction pathways influenced by induced cellular transformation of NPM-ALK.
- Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003 Mar 13;422(6928):198-207.
- Crockett DK, Lin Z, Elenitoba-Johnson KS and Lim MS Identification of NPM-ALK interacting proteins by tandem mass spectrometry. Oncogene 2004 In press.
- Duyster J, Bai RY, Morris SW. Translocations involving anaplastic lymphoma kinase (ALK). Oncogene. 2001 Sep 10;20(40):5623-37.
- Edmondson RD, Vondriska TM, Biederman KJ, Zhang J, Jones RC, Zheng Y, Allen DL, Xiu JX, Cardwell EM, Pisano MR, Ping P. Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS. Mol Cell Proteomics. 2002 Jun;1(6):421-33.
- Figeys D, McBroom LD, Moran MF.Mass spectrometry for the study of protein-protein interactions. Methods. 2001 Jul;24(3):230-9.
- Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags Nat Biotechnol. 1999 Oct;17(10):994-9.
- Kadin ME, Carpenter C. Systemic and primary cutaneous anaplastic large cell lymphomas. Semin Hematol. 2003 Jul;40(3):244-56.
- Morris SW, Xue L, Ma Z, Kinney MC. Alk+ CD30+ lymphomas: a distinct molecular genetic subtype of non-Hodgkin's lymphoma. Br J Haematol. 2001 May;113(2):275-95.
- Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL and Look AT (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non- Hodgkin's lymphoma Science, 263, 1281-4.
- Shiio Y, Donohoe S, Yi EC, Goodlett DR, Aebersold R, Eisenman RN.Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 2002 Oct 1;21(19):5088-96.