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Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development.

Colognato H, Ramachandrappa S, Olsen IM, ffrench-Constant C - J. Cell Biol. (2004)

Bottom Line: Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling.Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation).However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge CB21QP, England, UK. colognato@pharm.sunysb.edu

ABSTRACT
Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.

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SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.
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fig8: SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.

Mentions: We asked whether ligand binding by each integrin specifically altered associated SFK activity. We tested this using antibodies specific for two phosphorylated tyrosines within Src that also recognize the following conserved tyrosine (Y) sites in Fyn and Lyn: (1) Y418 (Y420 and Y397 in rat Fyn and Lyn, respectively), which is an autophosphorylation site; and (2) Y527 (Y531 and Y508 in rat Fyn and Lyn, respectively), which is phosphorylated by COOH-terminal Src kinase (Csk) to inactivate the SFK. First, we used the antibody against Src phosphoY418 to examine the phosphorylation of Fyn or Lyn by oligodendrocyte precursors grown on PDL, Lm2, or FN (Fig. 8 A). We found that the catalytic tyrosine was phosphorylated on Fyn, independent of substrate. In contrast, on Lyn, phosphorylation of Y397 was only seen in cells grown on the αVβ3 ligand FN, suggesting that this SFK was activated by αVβ3 and not by α6β1.


Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development.

Colognato H, Ramachandrappa S, Olsen IM, ffrench-Constant C - J. Cell Biol. (2004)

SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.
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Related In: Results  -  Collection

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fig8: SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.
Mentions: We asked whether ligand binding by each integrin specifically altered associated SFK activity. We tested this using antibodies specific for two phosphorylated tyrosines within Src that also recognize the following conserved tyrosine (Y) sites in Fyn and Lyn: (1) Y418 (Y420 and Y397 in rat Fyn and Lyn, respectively), which is an autophosphorylation site; and (2) Y527 (Y531 and Y508 in rat Fyn and Lyn, respectively), which is phosphorylated by COOH-terminal Src kinase (Csk) to inactivate the SFK. First, we used the antibody against Src phosphoY418 to examine the phosphorylation of Fyn or Lyn by oligodendrocyte precursors grown on PDL, Lm2, or FN (Fig. 8 A). We found that the catalytic tyrosine was phosphorylated on Fyn, independent of substrate. In contrast, on Lyn, phosphorylation of Y397 was only seen in cells grown on the αVβ3 ligand FN, suggesting that this SFK was activated by αVβ3 and not by α6β1.

Bottom Line: Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling.Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation).However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge CB21QP, England, UK. colognato@pharm.sunysb.edu

ABSTRACT
Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.

Show MeSH
Related in: MedlinePlus