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Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

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Phosphorylation of CRMP2, a GSK-3β substrate, in transgenic mice. (A) Brain cytosol fractions were probed with antibody 3F4, which recognizes CRMP2 phosphorylated at T509 and S522. Note the elevated levels of phospho-CRMP2 in FeCγ transgenic mice compared with Fe.27 or nontransgenic control mice. (B). The blot used above was stripped and reprobed with antibody C4G, which recognizes total CRMP2. The levels of total CRMP2 are not changed. (C) Quantitative analysis of phospho-CRMP2 levels as detected by 3F4 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (D) Levels of CRMP2 phosphorylated at T514 were not changed in transgenic mice (top panel). The blot was stripped and reprobed with antitubulin antibodies as a loading control (bottom panel). (E) A hypothetical cascade of signaling events, similar to Sema3a signaling pathway, suggests a role for APP in axonal guidance. The cleavage of APP to release AICD activates GSK-3β and results in phosphorylation of CRMP-2 at S522 and T509–the same residues that mediate the repulsive action of Sema3a upon binding to Plexin/Neuropilin receptors. F-spondin, an extracellular signaling protein of floor plate and hippocampus which is involved in axonal pathfinding and neurite outgrowth, could be a candidate signaling protein (shown by “?”) because it binds APP and inhibits AICD production.
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fig6: Phosphorylation of CRMP2, a GSK-3β substrate, in transgenic mice. (A) Brain cytosol fractions were probed with antibody 3F4, which recognizes CRMP2 phosphorylated at T509 and S522. Note the elevated levels of phospho-CRMP2 in FeCγ transgenic mice compared with Fe.27 or nontransgenic control mice. (B). The blot used above was stripped and reprobed with antibody C4G, which recognizes total CRMP2. The levels of total CRMP2 are not changed. (C) Quantitative analysis of phospho-CRMP2 levels as detected by 3F4 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (D) Levels of CRMP2 phosphorylated at T514 were not changed in transgenic mice (top panel). The blot was stripped and reprobed with antitubulin antibodies as a loading control (bottom panel). (E) A hypothetical cascade of signaling events, similar to Sema3a signaling pathway, suggests a role for APP in axonal guidance. The cleavage of APP to release AICD activates GSK-3β and results in phosphorylation of CRMP-2 at S522 and T509–the same residues that mediate the repulsive action of Sema3a upon binding to Plexin/Neuropilin receptors. F-spondin, an extracellular signaling protein of floor plate and hippocampus which is involved in axonal pathfinding and neurite outgrowth, could be a candidate signaling protein (shown by “?”) because it binds APP and inhibits AICD production.

Mentions: Many proteins are phosphorylated by GSK-3β on multiple Ser/Thr residues; the microtubule binding proteins, tau, microtubule-associated protein 1B, and collapsin responsive mediator protein–2 (CRMP2), are among the known GSK-3 substrates. These proteins bind and stimulate microtubule stability, but they fail to bind microtubules upon phosphorylation and cause microtubule depolymerization (Fukata et al., 2002; Jope and Johnson, 2004). We examined the status of CRMP2, a neuronal-specific protein that is involved in axonal repulsion and neuronal polarity, in our transgenic mice. CRMP2 mediates the repulsive action of Semaphorin3a (Sema3a), which binds the Plexin receptors in growth cones, activates GSK-3β, and phosphorylates CRMP2 on T509 and S522 (Uchida et al., 2005). An antibody, 3F4, which originally was raised against the neurofibrillary tangles (NFTs) from AD brains (Yoshida et al., 1998; Gu et al., 2000), specifically recognizes CRMP2 phosphorylated at T509 and S522 (Uchida et al., 2005). We examined the phosphorylation status of CRMP2 in our FeCγ transgenic mice by using the following antibodies: antibody 3F4 to detect CRMP2 phosphorylated at T509 and S522 (Uchida et al., 2005); antibody pT514 to recognize CRMP2 phosphorylated at T514 (Yoshimura et al., 2005); and antibody C4G that recognizes total CRMP2 (Gu et al., 2000). We immunoblotted brain cytosolic fractions from the indicated mice with antibody 3F4, and observed that the levels of phosphoCRMP2 were significantly higher in FeCγ.12 and FeCγ.25 animals compared with nontransgenic control or Fe.27 mice (Fig. 6 A). We stripped the blot and reprobed with antibody C4G that recognizes total CRMP2, and found that the total levels of CRMP2 were unaltered (Fig. 6 B). Quantification of the protein levels showed a two- to threefold increase in phosho-CRMP2 in the AICD transgenic mice compared with Fe.27 or normal mice (Fig. 6 C)


Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

Phosphorylation of CRMP2, a GSK-3β substrate, in transgenic mice. (A) Brain cytosol fractions were probed with antibody 3F4, which recognizes CRMP2 phosphorylated at T509 and S522. Note the elevated levels of phospho-CRMP2 in FeCγ transgenic mice compared with Fe.27 or nontransgenic control mice. (B). The blot used above was stripped and reprobed with antibody C4G, which recognizes total CRMP2. The levels of total CRMP2 are not changed. (C) Quantitative analysis of phospho-CRMP2 levels as detected by 3F4 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (D) Levels of CRMP2 phosphorylated at T514 were not changed in transgenic mice (top panel). The blot was stripped and reprobed with antitubulin antibodies as a loading control (bottom panel). (E) A hypothetical cascade of signaling events, similar to Sema3a signaling pathway, suggests a role for APP in axonal guidance. The cleavage of APP to release AICD activates GSK-3β and results in phosphorylation of CRMP-2 at S522 and T509–the same residues that mediate the repulsive action of Sema3a upon binding to Plexin/Neuropilin receptors. F-spondin, an extracellular signaling protein of floor plate and hippocampus which is involved in axonal pathfinding and neurite outgrowth, could be a candidate signaling protein (shown by “?”) because it binds APP and inhibits AICD production.
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Related In: Results  -  Collection

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fig6: Phosphorylation of CRMP2, a GSK-3β substrate, in transgenic mice. (A) Brain cytosol fractions were probed with antibody 3F4, which recognizes CRMP2 phosphorylated at T509 and S522. Note the elevated levels of phospho-CRMP2 in FeCγ transgenic mice compared with Fe.27 or nontransgenic control mice. (B). The blot used above was stripped and reprobed with antibody C4G, which recognizes total CRMP2. The levels of total CRMP2 are not changed. (C) Quantitative analysis of phospho-CRMP2 levels as detected by 3F4 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (D) Levels of CRMP2 phosphorylated at T514 were not changed in transgenic mice (top panel). The blot was stripped and reprobed with antitubulin antibodies as a loading control (bottom panel). (E) A hypothetical cascade of signaling events, similar to Sema3a signaling pathway, suggests a role for APP in axonal guidance. The cleavage of APP to release AICD activates GSK-3β and results in phosphorylation of CRMP-2 at S522 and T509–the same residues that mediate the repulsive action of Sema3a upon binding to Plexin/Neuropilin receptors. F-spondin, an extracellular signaling protein of floor plate and hippocampus which is involved in axonal pathfinding and neurite outgrowth, could be a candidate signaling protein (shown by “?”) because it binds APP and inhibits AICD production.
Mentions: Many proteins are phosphorylated by GSK-3β on multiple Ser/Thr residues; the microtubule binding proteins, tau, microtubule-associated protein 1B, and collapsin responsive mediator protein–2 (CRMP2), are among the known GSK-3 substrates. These proteins bind and stimulate microtubule stability, but they fail to bind microtubules upon phosphorylation and cause microtubule depolymerization (Fukata et al., 2002; Jope and Johnson, 2004). We examined the status of CRMP2, a neuronal-specific protein that is involved in axonal repulsion and neuronal polarity, in our transgenic mice. CRMP2 mediates the repulsive action of Semaphorin3a (Sema3a), which binds the Plexin receptors in growth cones, activates GSK-3β, and phosphorylates CRMP2 on T509 and S522 (Uchida et al., 2005). An antibody, 3F4, which originally was raised against the neurofibrillary tangles (NFTs) from AD brains (Yoshida et al., 1998; Gu et al., 2000), specifically recognizes CRMP2 phosphorylated at T509 and S522 (Uchida et al., 2005). We examined the phosphorylation status of CRMP2 in our FeCγ transgenic mice by using the following antibodies: antibody 3F4 to detect CRMP2 phosphorylated at T509 and S522 (Uchida et al., 2005); antibody pT514 to recognize CRMP2 phosphorylated at T514 (Yoshimura et al., 2005); and antibody C4G that recognizes total CRMP2 (Gu et al., 2000). We immunoblotted brain cytosolic fractions from the indicated mice with antibody 3F4, and observed that the levels of phosphoCRMP2 were significantly higher in FeCγ.12 and FeCγ.25 animals compared with nontransgenic control or Fe.27 mice (Fig. 6 A). We stripped the blot and reprobed with antibody C4G that recognizes total CRMP2, and found that the total levels of CRMP2 were unaltered (Fig. 6 B). Quantification of the protein levels showed a two- to threefold increase in phosho-CRMP2 in the AICD transgenic mice compared with Fe.27 or normal mice (Fig. 6 C)

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

Show MeSH
Related in: MedlinePlus