The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

Claudia Voena, Chiara Conte, Chiara Ambrogio, Elisabetta Boeri Erba, Francesco Boccalatte, Shabaz Mohammed, Ole N Jensen, Giorgio Palestro, Giorgio Inghirami, Roberto Chiarle

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.
Original languageEnglish
JournalCancer Research
Volume67
Issue number9
Pages (from-to)4278-4286
Number of pages8
ISSN0008-5472
DOIs
Publication statusPublished - 1. May 2007

Fingerprint

Phosphoric Monoester Hydrolases
Cell Movement
Tyrosine
Lymphoma
Anaplastic Large-Cell Lymphoma
Growth
Small Interfering RNA
Growth Factor Receptors
HEK293 Cells
Cell Proliferation
Cell Line
src Homology Domains
Mitogen-Activated Protein Kinase 1
Apoptosis

Keywords

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Apoptosis
  • Cell Growth Processes
  • Cell Movement
  • Down-Regulation
  • Enzyme Activation
  • GRB2 Adaptor Protein
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • K562 Cells
  • Lymphoma, Large B-Cell, Diffuse
  • Mass Spectrometry
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • Protein Tyrosine Phosphatases
  • Protein-Tyrosine Kinases
  • RNA, Small Interfering
  • SH2 Domain-Containing Protein Tyrosine Phosphatases
  • Transfection

Cite this

Voena, C., Conte, C., Ambrogio, C., Boeri Erba, E., Boccalatte, F., Mohammed, S., ... Chiarle, R. (2007). The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration. Cancer Research, 67(9), 4278-4286. https://doi.org/10.1158/0008-5472.CAN-06-4350
Voena, Claudia ; Conte, Chiara ; Ambrogio, Chiara ; Boeri Erba, Elisabetta ; Boccalatte, Francesco ; Mohammed, Shabaz ; Jensen, Ole N ; Palestro, Giorgio ; Inghirami, Giorgio ; Chiarle, Roberto. / The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration. In: Cancer Research. 2007 ; Vol. 67, No. 9. pp. 4278-4286.
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Voena, C, Conte, C, Ambrogio, C, Boeri Erba, E, Boccalatte, F, Mohammed, S, Jensen, ON, Palestro, G, Inghirami, G & Chiarle, R 2007, 'The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration', Cancer Research, vol. 67, no. 9, pp. 4278-4286. https://doi.org/10.1158/0008-5472.CAN-06-4350

The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration. / Voena, Claudia; Conte, Chiara; Ambrogio, Chiara; Boeri Erba, Elisabetta; Boccalatte, Francesco; Mohammed, Shabaz; Jensen, Ole N; Palestro, Giorgio; Inghirami, Giorgio; Chiarle, Roberto.

In: Cancer Research, Vol. 67, No. 9, 01.05.2007, p. 4278-4286.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

AU - Voena, Claudia

AU - Conte, Chiara

AU - Ambrogio, Chiara

AU - Boeri Erba, Elisabetta

AU - Boccalatte, Francesco

AU - Mohammed, Shabaz

AU - Jensen, Ole N

AU - Palestro, Giorgio

AU - Inghirami, Giorgio

AU - Chiarle, Roberto

PY - 2007/5/1

Y1 - 2007/5/1

N2 - Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

AB - Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

KW - Adaptor Proteins, Signal Transducing

KW - Amino Acid Sequence

KW - Apoptosis

KW - Cell Growth Processes

KW - Cell Movement

KW - Down-Regulation

KW - Enzyme Activation

KW - GRB2 Adaptor Protein

KW - Humans

KW - Intracellular Signaling Peptides and Proteins

KW - K562 Cells

KW - Lymphoma, Large B-Cell, Diffuse

KW - Mass Spectrometry

KW - Molecular Sequence Data

KW - Phosphorylation

KW - Protein Tyrosine Phosphatase, Non-Receptor Type 11

KW - Protein Tyrosine Phosphatases

KW - Protein-Tyrosine Kinases

KW - RNA, Small Interfering

KW - SH2 Domain-Containing Protein Tyrosine Phosphatases

KW - Transfection

U2 - 10.1158/0008-5472.CAN-06-4350

DO - 10.1158/0008-5472.CAN-06-4350

M3 - Journal article

VL - 67

SP - 4278

EP - 4286

JO - Cancer Research

JF - Cancer Research

SN - 0008-5472

IS - 9

ER -