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Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

Ahmed Z, Timsah Z, Suen KM, Cook NP, Lee GR, Lin CC, Gagea M, Marti AA, Ladbury JE - Nat Commun (2015)

Bottom Line: Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Center for Biomolecular Structure and Function, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

No MeSH data available.


Related in: MedlinePlus

Analysis of human colon and prostate cancer tissues for Y160 phosphorylation.Using the pY160 antibody IHC was performed on multi-tumours tissue microarray of 95 samples with 40 types of tumours from 27 organs. (a) Weak or no staining for Grb2 Y160 phosphorylation in grade I colon adenocarcinoma. (b) Strong staining for Grb2 Y160 phosphorylation in grade III colon adenocarcinoma. (c) Weak or no staining for Grb2 Y160 phosphorylation in grade II prostate adenocarcinoma. (d) Moderate staining for Grb2 Y160 phosphorylation in grade III prostate adenocarcinoma. A significant increase in the level of pY160 phosphorylation is seen in higher-grade tumour samples. The bars on a–d correspond to 50 μm. (e) The pY160 antibody staining patterns for 118 colon cancer tissue samples. The samples were scored according to the pY160 staining as weak or none, moderate and strong and plotted against tumour grade as percentage. Data compiled from normal tissues (n=42), and tumours grade I (n=28), grade II (n=34) and grade III (n=14) which shows a progressive increase in the strength of pY160 staining with higher tumour grade. (f) The pY160 antibody staining patterns for 42 clinical prostate cancer tissue samples with the relative staining patterns for pY160 antibody. As above, samples were scored, sorted and plotted against tumour grade as percentage. Normal and/or hyperplasic tissue (n=15), tumour grade II (n=13) and grade III (n=14). Here the pY160 staining is only associated with malignant tumours and intensity of staining is increased in tumours with a higher level of malignancy.
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f5: Analysis of human colon and prostate cancer tissues for Y160 phosphorylation.Using the pY160 antibody IHC was performed on multi-tumours tissue microarray of 95 samples with 40 types of tumours from 27 organs. (a) Weak or no staining for Grb2 Y160 phosphorylation in grade I colon adenocarcinoma. (b) Strong staining for Grb2 Y160 phosphorylation in grade III colon adenocarcinoma. (c) Weak or no staining for Grb2 Y160 phosphorylation in grade II prostate adenocarcinoma. (d) Moderate staining for Grb2 Y160 phosphorylation in grade III prostate adenocarcinoma. A significant increase in the level of pY160 phosphorylation is seen in higher-grade tumour samples. The bars on a–d correspond to 50 μm. (e) The pY160 antibody staining patterns for 118 colon cancer tissue samples. The samples were scored according to the pY160 staining as weak or none, moderate and strong and plotted against tumour grade as percentage. Data compiled from normal tissues (n=42), and tumours grade I (n=28), grade II (n=34) and grade III (n=14) which shows a progressive increase in the strength of pY160 staining with higher tumour grade. (f) The pY160 antibody staining patterns for 42 clinical prostate cancer tissue samples with the relative staining patterns for pY160 antibody. As above, samples were scored, sorted and plotted against tumour grade as percentage. Normal and/or hyperplasic tissue (n=15), tumour grade II (n=13) and grade III (n=14). Here the pY160 staining is only associated with malignant tumours and intensity of staining is increased in tumours with a higher level of malignancy.

Mentions: Y160 is a target phosphorylation site for many receptor and non-receptor tyrosine kinases that are involved in cancer growth and metastasis21. This suggests dGrb2 may have an inhibitory role whilst mGrb2 promotes proliferative downstream signalling. Here we have shown that Y160 phosphorylation-dependent dissociation of dGrb2 is sufficient to induce upregulation of MAPK signalling (Fig. 4) which is associated with proliferation and metastasis232425. Therefore, we would predict that in tyrosine kinase-driven cancers phosphorylation of Y160 and the resulting dissociation of Grb2 dimers would be capable of inducing cell proliferative and metastatic outcome. This would suggest that pY160 could be used as marker for elevated tyrosine kinase-mediated ERK signalling in human cancers. We therefore generated a site-specific anti-pY160 antibody against pY160. This antibody was tested exhaustively and found to be suitable for detecting Y160-phosphorylated Grb2 in western blotting and in immunohistochemistry (IHC) analysis (Supplementary Fig. 7). We performed IHC analysis of a multi-tumour tissue array containing 40 human tumour samples from 27 different organs. The results show that Y160 was highly phosphorylated in a significant number of high-grade malignant tumours but not in the lower grade or non-malignant counterpart tumours or in corresponding normal tissue (Supplementary Fig. 8). Y160 phosphorylation was particularly prevalent in higher grade, late-stage malignant tumours of the colon and prostate (Fig. 5a–d). Therefore, we performed additional IHC analysis on 118 colon and 42 prostate cancer tissues with the anti-pY160 antibody, which shows a clear correlation between disease progression and Grb2 phosphorylation (Fig. 5e,f). Higher levels of Y160 phosphorylation were again clearly associated with higher-grade malignant tumours. In addition we performed protein microarray analysis on 55 human breast tumours with matched normal tissue using the pY160 antibody. The results revealed Y160 phosphorylation was absent in normal breast samples, however 12 out of 55 breast tumour samples were positive for pY160 (Supplementary Fig. 9a–c). To confirm the results of the protein microarray, we selected two positive breast tumour samples and the corresponding normal matched sample for western blotting analysis. The results show elevated Grb2 expression and phosphorylation, MAP kinase and tyrosine kinase activity in the tumour samples compared with the normal cohort. Furthermore, it was possible to assign the elevated pY160 level of one patient to increased EGFR expression and kinase activity (Supplementary Fig. 9d).


Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

Ahmed Z, Timsah Z, Suen KM, Cook NP, Lee GR, Lin CC, Gagea M, Marti AA, Ladbury JE - Nat Commun (2015)

Analysis of human colon and prostate cancer tissues for Y160 phosphorylation.Using the pY160 antibody IHC was performed on multi-tumours tissue microarray of 95 samples with 40 types of tumours from 27 organs. (a) Weak or no staining for Grb2 Y160 phosphorylation in grade I colon adenocarcinoma. (b) Strong staining for Grb2 Y160 phosphorylation in grade III colon adenocarcinoma. (c) Weak or no staining for Grb2 Y160 phosphorylation in grade II prostate adenocarcinoma. (d) Moderate staining for Grb2 Y160 phosphorylation in grade III prostate adenocarcinoma. A significant increase in the level of pY160 phosphorylation is seen in higher-grade tumour samples. The bars on a–d correspond to 50 μm. (e) The pY160 antibody staining patterns for 118 colon cancer tissue samples. The samples were scored according to the pY160 staining as weak or none, moderate and strong and plotted against tumour grade as percentage. Data compiled from normal tissues (n=42), and tumours grade I (n=28), grade II (n=34) and grade III (n=14) which shows a progressive increase in the strength of pY160 staining with higher tumour grade. (f) The pY160 antibody staining patterns for 42 clinical prostate cancer tissue samples with the relative staining patterns for pY160 antibody. As above, samples were scored, sorted and plotted against tumour grade as percentage. Normal and/or hyperplasic tissue (n=15), tumour grade II (n=13) and grade III (n=14). Here the pY160 staining is only associated with malignant tumours and intensity of staining is increased in tumours with a higher level of malignancy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491180&req=5

f5: Analysis of human colon and prostate cancer tissues for Y160 phosphorylation.Using the pY160 antibody IHC was performed on multi-tumours tissue microarray of 95 samples with 40 types of tumours from 27 organs. (a) Weak or no staining for Grb2 Y160 phosphorylation in grade I colon adenocarcinoma. (b) Strong staining for Grb2 Y160 phosphorylation in grade III colon adenocarcinoma. (c) Weak or no staining for Grb2 Y160 phosphorylation in grade II prostate adenocarcinoma. (d) Moderate staining for Grb2 Y160 phosphorylation in grade III prostate adenocarcinoma. A significant increase in the level of pY160 phosphorylation is seen in higher-grade tumour samples. The bars on a–d correspond to 50 μm. (e) The pY160 antibody staining patterns for 118 colon cancer tissue samples. The samples were scored according to the pY160 staining as weak or none, moderate and strong and plotted against tumour grade as percentage. Data compiled from normal tissues (n=42), and tumours grade I (n=28), grade II (n=34) and grade III (n=14) which shows a progressive increase in the strength of pY160 staining with higher tumour grade. (f) The pY160 antibody staining patterns for 42 clinical prostate cancer tissue samples with the relative staining patterns for pY160 antibody. As above, samples were scored, sorted and plotted against tumour grade as percentage. Normal and/or hyperplasic tissue (n=15), tumour grade II (n=13) and grade III (n=14). Here the pY160 staining is only associated with malignant tumours and intensity of staining is increased in tumours with a higher level of malignancy.
Mentions: Y160 is a target phosphorylation site for many receptor and non-receptor tyrosine kinases that are involved in cancer growth and metastasis21. This suggests dGrb2 may have an inhibitory role whilst mGrb2 promotes proliferative downstream signalling. Here we have shown that Y160 phosphorylation-dependent dissociation of dGrb2 is sufficient to induce upregulation of MAPK signalling (Fig. 4) which is associated with proliferation and metastasis232425. Therefore, we would predict that in tyrosine kinase-driven cancers phosphorylation of Y160 and the resulting dissociation of Grb2 dimers would be capable of inducing cell proliferative and metastatic outcome. This would suggest that pY160 could be used as marker for elevated tyrosine kinase-mediated ERK signalling in human cancers. We therefore generated a site-specific anti-pY160 antibody against pY160. This antibody was tested exhaustively and found to be suitable for detecting Y160-phosphorylated Grb2 in western blotting and in immunohistochemistry (IHC) analysis (Supplementary Fig. 7). We performed IHC analysis of a multi-tumour tissue array containing 40 human tumour samples from 27 different organs. The results show that Y160 was highly phosphorylated in a significant number of high-grade malignant tumours but not in the lower grade or non-malignant counterpart tumours or in corresponding normal tissue (Supplementary Fig. 8). Y160 phosphorylation was particularly prevalent in higher grade, late-stage malignant tumours of the colon and prostate (Fig. 5a–d). Therefore, we performed additional IHC analysis on 118 colon and 42 prostate cancer tissues with the anti-pY160 antibody, which shows a clear correlation between disease progression and Grb2 phosphorylation (Fig. 5e,f). Higher levels of Y160 phosphorylation were again clearly associated with higher-grade malignant tumours. In addition we performed protein microarray analysis on 55 human breast tumours with matched normal tissue using the pY160 antibody. The results revealed Y160 phosphorylation was absent in normal breast samples, however 12 out of 55 breast tumour samples were positive for pY160 (Supplementary Fig. 9a–c). To confirm the results of the protein microarray, we selected two positive breast tumour samples and the corresponding normal matched sample for western blotting analysis. The results show elevated Grb2 expression and phosphorylation, MAP kinase and tyrosine kinase activity in the tumour samples compared with the normal cohort. Furthermore, it was possible to assign the elevated pY160 level of one patient to increased EGFR expression and kinase activity (Supplementary Fig. 9d).

Bottom Line: Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA [2] Center for Biomolecular Structure and Function, University of Texas, M.D. Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

ABSTRACT
The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

No MeSH data available.


Related in: MedlinePlus