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Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

Palacios-Moreno J, Foltz L, Guo A, Stokes MP, Kuehn ED, George L, Comb M, Grimes ML - PLoS Comput. Biol. (2015)

Bottom Line: Clusters of proteins in these networks are indicative of functional signaling pathways.The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN.Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, Center for Structural and Functional Neuroscience, University of Montana, Missoula, Montana, United States of America.

ABSTRACT
Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

No MeSH data available.


Related in: MedlinePlus

Fold change in response to RTK stimulation or inhibition.Shown are changes of more than twofold from representative experiments where peak intensity was measured for treatment and control conditions in the same experiment with cell lines and treatments indicated on column labels (e.g., “NGF to C” means NGF-treated compared to control). (A) Total phosphorylation changes in tyrosine kinases. SFKs phosphorylated on their C-terminal inhibitory site were tracked separately (SFK_i). In addition to results summarized in the text, the ALK inhibitor, TAE684, inhibited RET and IGF1R phosphorylation about threefold in SH-SY5Y cells. NGF stimulated phosphorylation of IGF1R and PDGFRA, and BDNF treatment increased phosphorylation of FGFR1, in SMS-KCN cells. EGFR and EPHA2 were affected in opposite ways in LAN-6 and SH-SY5Y cells. AXL, PDGFRB, EPHA7, and EPHB1 phosphorylation were decreased by NGF in LAN6 cells. Individual phosphorylation site changes are shown for SFKs (B). Activating (SFK Y411-426) and inhibitory (SFK Y508-531) sites on FYN, LYN, YES1, and SRC were affected differently by different treatments. Phosphorylation sites represent the sum of all peptides surrounding that site; peptides whose conserved sequence is present in several proteins are indicated with multiple names, e.g., “FYN 420; LCK 394; SRC 419; YES1 426.” Fold changes are graphed on a blue-yellow color scale with blue representing a decrease, and yellow, an increase, compared to control (key). Data are sorted from most to least for each total row (protein or phosphorylation site) and column (treatment) from left to right and top to bottom, respectively.
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pcbi.1004130.g004: Fold change in response to RTK stimulation or inhibition.Shown are changes of more than twofold from representative experiments where peak intensity was measured for treatment and control conditions in the same experiment with cell lines and treatments indicated on column labels (e.g., “NGF to C” means NGF-treated compared to control). (A) Total phosphorylation changes in tyrosine kinases. SFKs phosphorylated on their C-terminal inhibitory site were tracked separately (SFK_i). In addition to results summarized in the text, the ALK inhibitor, TAE684, inhibited RET and IGF1R phosphorylation about threefold in SH-SY5Y cells. NGF stimulated phosphorylation of IGF1R and PDGFRA, and BDNF treatment increased phosphorylation of FGFR1, in SMS-KCN cells. EGFR and EPHA2 were affected in opposite ways in LAN-6 and SH-SY5Y cells. AXL, PDGFRB, EPHA7, and EPHB1 phosphorylation were decreased by NGF in LAN6 cells. Individual phosphorylation site changes are shown for SFKs (B). Activating (SFK Y411-426) and inhibitory (SFK Y508-531) sites on FYN, LYN, YES1, and SRC were affected differently by different treatments. Phosphorylation sites represent the sum of all peptides surrounding that site; peptides whose conserved sequence is present in several proteins are indicated with multiple names, e.g., “FYN 420; LCK 394; SRC 419; YES1 426.” Fold changes are graphed on a blue-yellow color scale with blue representing a decrease, and yellow, an increase, compared to control (key). Data are sorted from most to least for each total row (protein or phosphorylation site) and column (treatment) from left to right and top to bottom, respectively.

Mentions: RTK activation affects other RTKs, SFKs, and other tyrosine kinases. To examine the effects of RTK stimulation on other tyrosine kinases, we compared phosphoproteomic data from cells treated to influence RTK activity, or not treated, in the same experiment. Fig 4A shows tyrosine kinases whose total phosphorylation changed more than two-fold under experimental conditions where RTKs were stimulated by ligand or ALK was inhibited. For example, NGF treatment caused a more than twofold increase in total phosphorylation of DDR2, and more than fivefold decrease in phosphorylation of PDGFRA in both LAN-6 and SH-SY5Y cells. EGF treatment of SK-N-BE(2) cells activated EGFR and stimulated EPHA3 phosphorylation about 3-fold (Fig 4A). These data indicate that stimulation of one RTK affects the phosphorylation state of other RTKs in neuroblastoma cell lines.


Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

Palacios-Moreno J, Foltz L, Guo A, Stokes MP, Kuehn ED, George L, Comb M, Grimes ML - PLoS Comput. Biol. (2015)

Fold change in response to RTK stimulation or inhibition.Shown are changes of more than twofold from representative experiments where peak intensity was measured for treatment and control conditions in the same experiment with cell lines and treatments indicated on column labels (e.g., “NGF to C” means NGF-treated compared to control). (A) Total phosphorylation changes in tyrosine kinases. SFKs phosphorylated on their C-terminal inhibitory site were tracked separately (SFK_i). In addition to results summarized in the text, the ALK inhibitor, TAE684, inhibited RET and IGF1R phosphorylation about threefold in SH-SY5Y cells. NGF stimulated phosphorylation of IGF1R and PDGFRA, and BDNF treatment increased phosphorylation of FGFR1, in SMS-KCN cells. EGFR and EPHA2 were affected in opposite ways in LAN-6 and SH-SY5Y cells. AXL, PDGFRB, EPHA7, and EPHB1 phosphorylation were decreased by NGF in LAN6 cells. Individual phosphorylation site changes are shown for SFKs (B). Activating (SFK Y411-426) and inhibitory (SFK Y508-531) sites on FYN, LYN, YES1, and SRC were affected differently by different treatments. Phosphorylation sites represent the sum of all peptides surrounding that site; peptides whose conserved sequence is present in several proteins are indicated with multiple names, e.g., “FYN 420; LCK 394; SRC 419; YES1 426.” Fold changes are graphed on a blue-yellow color scale with blue representing a decrease, and yellow, an increase, compared to control (key). Data are sorted from most to least for each total row (protein or phosphorylation site) and column (treatment) from left to right and top to bottom, respectively.
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Related In: Results  -  Collection

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pcbi.1004130.g004: Fold change in response to RTK stimulation or inhibition.Shown are changes of more than twofold from representative experiments where peak intensity was measured for treatment and control conditions in the same experiment with cell lines and treatments indicated on column labels (e.g., “NGF to C” means NGF-treated compared to control). (A) Total phosphorylation changes in tyrosine kinases. SFKs phosphorylated on their C-terminal inhibitory site were tracked separately (SFK_i). In addition to results summarized in the text, the ALK inhibitor, TAE684, inhibited RET and IGF1R phosphorylation about threefold in SH-SY5Y cells. NGF stimulated phosphorylation of IGF1R and PDGFRA, and BDNF treatment increased phosphorylation of FGFR1, in SMS-KCN cells. EGFR and EPHA2 were affected in opposite ways in LAN-6 and SH-SY5Y cells. AXL, PDGFRB, EPHA7, and EPHB1 phosphorylation were decreased by NGF in LAN6 cells. Individual phosphorylation site changes are shown for SFKs (B). Activating (SFK Y411-426) and inhibitory (SFK Y508-531) sites on FYN, LYN, YES1, and SRC were affected differently by different treatments. Phosphorylation sites represent the sum of all peptides surrounding that site; peptides whose conserved sequence is present in several proteins are indicated with multiple names, e.g., “FYN 420; LCK 394; SRC 419; YES1 426.” Fold changes are graphed on a blue-yellow color scale with blue representing a decrease, and yellow, an increase, compared to control (key). Data are sorted from most to least for each total row (protein or phosphorylation site) and column (treatment) from left to right and top to bottom, respectively.
Mentions: RTK activation affects other RTKs, SFKs, and other tyrosine kinases. To examine the effects of RTK stimulation on other tyrosine kinases, we compared phosphoproteomic data from cells treated to influence RTK activity, or not treated, in the same experiment. Fig 4A shows tyrosine kinases whose total phosphorylation changed more than two-fold under experimental conditions where RTKs were stimulated by ligand or ALK was inhibited. For example, NGF treatment caused a more than twofold increase in total phosphorylation of DDR2, and more than fivefold decrease in phosphorylation of PDGFRA in both LAN-6 and SH-SY5Y cells. EGF treatment of SK-N-BE(2) cells activated EGFR and stimulated EPHA3 phosphorylation about 3-fold (Fig 4A). These data indicate that stimulation of one RTK affects the phosphorylation state of other RTKs in neuroblastoma cell lines.

Bottom Line: Clusters of proteins in these networks are indicative of functional signaling pathways.The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN.Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, Center for Structural and Functional Neuroscience, University of Montana, Missoula, Montana, United States of America.

ABSTRACT
Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

No MeSH data available.


Related in: MedlinePlus