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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

Intracellular localization of FYN and LYN changed in response to PTN and SCF.(A-D) Velocity gradient fractionation of intracellular organelles after serum starvation (control; squares) or 60 min stimulation of LAN-6 cells with PTN (A, C) or SCF (B, D). Data were quantified from western blots using antibodies against FYN (A, B) and LYN (C, D) and expressed as the percent of each protein in each gradient fraction after quantifying amounts in all other cell fractions (percent in whole cell). Shown are means from 2–4 experiments for each condition; error bars are SEM. (E) Organelle fractions, defined as pools of velocity gradient fractions lys, E1, E2, E3, and cyt as shown in (C), were subjected to flotation equilibrium centrifugation [32]. Western blots show these fractions and detergent-resistant (DRM) and-soluble (P1M) fractions (see S10 Fig) after no treatment (C = control) or treatment with PTN, probed with antibodies to FYN and LYN (indicated). Both floating (F) and non-floating (NF, defined as material in higher density fractions at the bottom of flotation equilibrium gradients) membranes were analyzed. That SFKs associated with floating (F) fractions indicates that they were robustly bound to membranes. (F) Phospho-SFK (left) and non-phospho-SFK antibodies (right) were used to immunoprecipitate proteins from endosome E1 fractions under unstimulated or stimulated conditions as in (A). Western blots were probed with FYN- and LYN-specific antibodies (indicated). (G) Box plot shows amounts of FYN (red) and LYN (blue) in detergent-resistant (DRM) and-soluble (P1M) fractions under control, ALK- or KIT-stimulated conditions as in A-D. (H) Bar plots show fold change (treatment/control if positive;-(treatment/control)-1 if negative) in all cell fractions under unstimulated or stimulated conditions as in A-D. (G, H) Amounts of FYN and LYN in all cell fractions were quantified from 3–7 experiments; boxes show quartiles and whiskers show ranges in G, error bars are SEM in H.
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pcbi.1004130.g007: Intracellular localization of FYN and LYN changed in response to PTN and SCF.(A-D) Velocity gradient fractionation of intracellular organelles after serum starvation (control; squares) or 60 min stimulation of LAN-6 cells with PTN (A, C) or SCF (B, D). Data were quantified from western blots using antibodies against FYN (A, B) and LYN (C, D) and expressed as the percent of each protein in each gradient fraction after quantifying amounts in all other cell fractions (percent in whole cell). Shown are means from 2–4 experiments for each condition; error bars are SEM. (E) Organelle fractions, defined as pools of velocity gradient fractions lys, E1, E2, E3, and cyt as shown in (C), were subjected to flotation equilibrium centrifugation [32]. Western blots show these fractions and detergent-resistant (DRM) and-soluble (P1M) fractions (see S10 Fig) after no treatment (C = control) or treatment with PTN, probed with antibodies to FYN and LYN (indicated). Both floating (F) and non-floating (NF, defined as material in higher density fractions at the bottom of flotation equilibrium gradients) membranes were analyzed. That SFKs associated with floating (F) fractions indicates that they were robustly bound to membranes. (F) Phospho-SFK (left) and non-phospho-SFK antibodies (right) were used to immunoprecipitate proteins from endosome E1 fractions under unstimulated or stimulated conditions as in (A). Western blots were probed with FYN- and LYN-specific antibodies (indicated). (G) Box plot shows amounts of FYN (red) and LYN (blue) in detergent-resistant (DRM) and-soluble (P1M) fractions under control, ALK- or KIT-stimulated conditions as in A-D. (H) Bar plots show fold change (treatment/control if positive;-(treatment/control)-1 if negative) in all cell fractions under unstimulated or stimulated conditions as in A-D. (G, H) Amounts of FYN and LYN in all cell fractions were quantified from 3–7 experiments; boxes show quartiles and whiskers show ranges in G, error bars are SEM in H.

Mentions: These data suggest the hypothesis that stimulation of different RTKs should affect the activity and intracellular localization of FYN and LYN. We used a cell fractionation approach to assay intracellular localization after stimulation of ALK with PTN and KIT with SCF (Fig 7). Amounts of FYN and LYN increased with PTN and SCF treatment in organelles whose migration on velocity sedimentation gradients overlaps with Rab7 and acid phosphatase [32], markers for late endosomes and lysosomes (Fig 7A–7D, fractions 4–7). SCF also induced increases mainly in FYN localization to fractions 8–11 (Fig 7B and 7D), which contain endosomes marked by Rab4 and Rab5 [32]. LYN and FYN also increased in fractions 16–22 in response to both ligands (Fig 7A–7D). These fractions contain soluble, cytoplasmic proteins, and signaling particles, which were previously resolved on gradients centrifuged with greater force [52]. FYN and LYN were robustly associated with membranes that floated to the density of endosomes on floatation equilibrium gradients, and amounts increased in organelles of higher sedimentation velocity (E1) after PTN treatment (Fig 7E). Both FYN and LYN were predominantly phosphorylated on their activating sites in these membranes (Fig 7F). Differences between FYN and LYN localization to detergent-resistant and-soluble fractions were also observed. FYN’s response to PTN (enhanced DRM and diminished P1M association; Fig 7G) was different from that to SCF (reduced DRM, enhanced P1M association). In contrast, LYN’s response to both ligands was similar (reduced DRM, increased P1M association; Fig 7G). The magnitude of ligand-induced changes in FYN and LYN in organelle fractions were distinct in response to PTN and SCF (Fig 7H). Increased FYN and LYN in faster sedimenting organelles (lys and E1 fractions) likely reflects migration to multivesicular bodies, late endosomes, and possibly lysosomes [32]. These data are consistent with the hypothesis that RTK activation regulates FYN and LYN localization and activity in neuroblastoma cells in a manner that distinguishes responses to individual RTKs.


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)

Intracellular localization of FYN and LYN changed in response to PTN and SCF.(A-D) Velocity gradient fractionation of intracellular organelles after serum starvation (control; squares) or 60 min stimulation of LAN-6 cells with PTN (A, C) or SCF (B, D). Data were quantified from western blots using antibodies against FYN (A, B) and LYN (C, D) and expressed as the percent of each protein in each gradient fraction after quantifying amounts in all other cell fractions (percent in whole cell). Shown are means from 2–4 experiments for each condition; error bars are SEM. (E) Organelle fractions, defined as pools of velocity gradient fractions lys, E1, E2, E3, and cyt as shown in (C), were subjected to flotation equilibrium centrifugation [32]. Western blots show these fractions and detergent-resistant (DRM) and-soluble (P1M) fractions (see S10 Fig) after no treatment (C = control) or treatment with PTN, probed with antibodies to FYN and LYN (indicated). Both floating (F) and non-floating (NF, defined as material in higher density fractions at the bottom of flotation equilibrium gradients) membranes were analyzed. That SFKs associated with floating (F) fractions indicates that they were robustly bound to membranes. (F) Phospho-SFK (left) and non-phospho-SFK antibodies (right) were used to immunoprecipitate proteins from endosome E1 fractions under unstimulated or stimulated conditions as in (A). Western blots were probed with FYN- and LYN-specific antibodies (indicated). (G) Box plot shows amounts of FYN (red) and LYN (blue) in detergent-resistant (DRM) and-soluble (P1M) fractions under control, ALK- or KIT-stimulated conditions as in A-D. (H) Bar plots show fold change (treatment/control if positive;-(treatment/control)-1 if negative) in all cell fractions under unstimulated or stimulated conditions as in A-D. (G, H) Amounts of FYN and LYN in all cell fractions were quantified from 3–7 experiments; boxes show quartiles and whiskers show ranges in G, error bars are SEM in H.
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pcbi.1004130.g007: Intracellular localization of FYN and LYN changed in response to PTN and SCF.(A-D) Velocity gradient fractionation of intracellular organelles after serum starvation (control; squares) or 60 min stimulation of LAN-6 cells with PTN (A, C) or SCF (B, D). Data were quantified from western blots using antibodies against FYN (A, B) and LYN (C, D) and expressed as the percent of each protein in each gradient fraction after quantifying amounts in all other cell fractions (percent in whole cell). Shown are means from 2–4 experiments for each condition; error bars are SEM. (E) Organelle fractions, defined as pools of velocity gradient fractions lys, E1, E2, E3, and cyt as shown in (C), were subjected to flotation equilibrium centrifugation [32]. Western blots show these fractions and detergent-resistant (DRM) and-soluble (P1M) fractions (see S10 Fig) after no treatment (C = control) or treatment with PTN, probed with antibodies to FYN and LYN (indicated). Both floating (F) and non-floating (NF, defined as material in higher density fractions at the bottom of flotation equilibrium gradients) membranes were analyzed. That SFKs associated with floating (F) fractions indicates that they were robustly bound to membranes. (F) Phospho-SFK (left) and non-phospho-SFK antibodies (right) were used to immunoprecipitate proteins from endosome E1 fractions under unstimulated or stimulated conditions as in (A). Western blots were probed with FYN- and LYN-specific antibodies (indicated). (G) Box plot shows amounts of FYN (red) and LYN (blue) in detergent-resistant (DRM) and-soluble (P1M) fractions under control, ALK- or KIT-stimulated conditions as in A-D. (H) Bar plots show fold change (treatment/control if positive;-(treatment/control)-1 if negative) in all cell fractions under unstimulated or stimulated conditions as in A-D. (G, H) Amounts of FYN and LYN in all cell fractions were quantified from 3–7 experiments; boxes show quartiles and whiskers show ranges in G, error bars are SEM in H.
Mentions: These data suggest the hypothesis that stimulation of different RTKs should affect the activity and intracellular localization of FYN and LYN. We used a cell fractionation approach to assay intracellular localization after stimulation of ALK with PTN and KIT with SCF (Fig 7). Amounts of FYN and LYN increased with PTN and SCF treatment in organelles whose migration on velocity sedimentation gradients overlaps with Rab7 and acid phosphatase [32], markers for late endosomes and lysosomes (Fig 7A–7D, fractions 4–7). SCF also induced increases mainly in FYN localization to fractions 8–11 (Fig 7B and 7D), which contain endosomes marked by Rab4 and Rab5 [32]. LYN and FYN also increased in fractions 16–22 in response to both ligands (Fig 7A–7D). These fractions contain soluble, cytoplasmic proteins, and signaling particles, which were previously resolved on gradients centrifuged with greater force [52]. FYN and LYN were robustly associated with membranes that floated to the density of endosomes on floatation equilibrium gradients, and amounts increased in organelles of higher sedimentation velocity (E1) after PTN treatment (Fig 7E). Both FYN and LYN were predominantly phosphorylated on their activating sites in these membranes (Fig 7F). Differences between FYN and LYN localization to detergent-resistant and-soluble fractions were also observed. FYN’s response to PTN (enhanced DRM and diminished P1M association; Fig 7G) was different from that to SCF (reduced DRM, enhanced P1M association). In contrast, LYN’s response to both ligands was similar (reduced DRM, increased P1M association; Fig 7G). The magnitude of ligand-induced changes in FYN and LYN in organelle fractions were distinct in response to PTN and SCF (Fig 7H). Increased FYN and LYN in faster sedimenting organelles (lys and E1 fractions) likely reflects migration to multivesicular bodies, late endosomes, and possibly lysosomes [32]. These data are consistent with the hypothesis that RTK activation regulates FYN and LYN localization and activity in neuroblastoma cells in a manner that distinguishes responses to individual RTKs.

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