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Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

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Expression of PAGFP-tagged GAP43 constructs in neuronally differentiated PC12 cells. (A) Schematic representation of the PAGFP-tagged GAP43 constructs. PAGFP with a farnesylation signal (PAGFP-F) and 3×PAGFP were prepared as reference constructs. PAGFP is indicated as a green box, the IQ domain (position 30–52 in rat GAP43) containing the phosphorylation site (Ser-41) as a red box, and the farnesylation signal, which directs lipidation of PAGFP-F, as a blue box. Right, calculated relative molecular weights. (B) Immunoblots of lysates of transfected PC12 cells expressing the respective constructs. Blots were developed using anti-GFP (left and right) and anti-GAP43 antibody (middle). GFP-tagged constructs are indicated by arrowheads, endogenous GAP43 by an arrow. The phosphomimicking GAP43 construct is not recognized by the anti-GAP43 antibody (JP91), suggesting that mutation of Ser-41 to Asp blocks binding of the antibody. Numbers to the sides of the blots indicate molecular mass standards in kilodaltons. (C) Fluorescence micrographs of PC12 cells transfected with the respective GAP43 constructs and controls. Cells were treated with NGF for 6 d. Exogenous proteins were detected using anti-GFP antibody. Note the enrichment of the GAP43 constructs in the tip of the processes. Scale bar, 50 μm.
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Figure 1: Expression of PAGFP-tagged GAP43 constructs in neuronally differentiated PC12 cells. (A) Schematic representation of the PAGFP-tagged GAP43 constructs. PAGFP with a farnesylation signal (PAGFP-F) and 3×PAGFP were prepared as reference constructs. PAGFP is indicated as a green box, the IQ domain (position 30–52 in rat GAP43) containing the phosphorylation site (Ser-41) as a red box, and the farnesylation signal, which directs lipidation of PAGFP-F, as a blue box. Right, calculated relative molecular weights. (B) Immunoblots of lysates of transfected PC12 cells expressing the respective constructs. Blots were developed using anti-GFP (left and right) and anti-GAP43 antibody (middle). GFP-tagged constructs are indicated by arrowheads, endogenous GAP43 by an arrow. The phosphomimicking GAP43 construct is not recognized by the anti-GAP43 antibody (JP91), suggesting that mutation of Ser-41 to Asp blocks binding of the antibody. Numbers to the sides of the blots indicate molecular mass standards in kilodaltons. (C) Fluorescence micrographs of PC12 cells transfected with the respective GAP43 constructs and controls. Cells were treated with NGF for 6 d. Exogenous proteins were detected using anti-GFP antibody. Note the enrichment of the GAP43 constructs in the tip of the processes. Scale bar, 50 μm.

Mentions: To generate a neuronal model to allow us to determine the dynamics and distribution of wild-type and modified GAP43 in living cells, we prepared a panel of constructs in which PAGFP was fused to GAP43wt and phosphoblocking (GAP43S41A) and phosphomimicking GAP43 mutants (GAP43S41D) at Ser-41 (Figure 1A). According to Simple Modular Architecture Research Tool (SMART) analysis (Schultz et al., 1998; Letunic et al., 2012), Ser-41 is located in an IQ domain, which serves as a binding site for EF-hand proteins such as calmodulin (Rhoads and Friedberg, 1997). For comparison, we used a cytosolic reference protein (3×PAGFP) and a PAGFP construct with a farnesylation signal from c-Ha-Ras fused to the C-terminus (PAGFP-F). Note that PAGFP-F most likely becomes also palmitoylated, since the sequence, which is fused to PAGFP, also contains palmitoylatable cysteine residues (Aronheim et al., 1994). Bioinformatic analysis revealed two potential palmitoylation sites in addition to the farnesylation site (CSS-Palm 2.0; Ren et al., 2008). Thus PAGFP-F could serve as a reference protein for membrane attachment, which requires at least dual lipidation (Shahinian and Silvius, 1995). The constructs were expressed in PC12 cells, which provide a well-established model for differentiating neurons. The GAP43 fusion proteins separated at an apparent molecular weight of ∼80 kDa (Figure 1B). The retarded mobility compared with the calculated molecular weight (52.6 kDa) is in agreement with the fact that GAP43 alone separates at higher molecular weight than calculated (Benowitz et al., 1987). 3×PAGFP separated at an apparent molecular weight of ∼95 kDa, which makes it appropriate as a cytosolic control construct of similar size. PAGFP-F separated at 32 kDa, close to the calculated molecular mass. We did not observe major degradation, indicating that the constructs are stable within the cells. Endogenous GAP43 and the GAP43wt and GAP43S41A fusion proteins were detected by an antibody against GAP43 (Figure 1B, middle, arrow). The antibody did not detect GAP43S41D, suggesting that the phosphomimicking mutation abolished antibody reactivity.


Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Expression of PAGFP-tagged GAP43 constructs in neuronally differentiated PC12 cells. (A) Schematic representation of the PAGFP-tagged GAP43 constructs. PAGFP with a farnesylation signal (PAGFP-F) and 3×PAGFP were prepared as reference constructs. PAGFP is indicated as a green box, the IQ domain (position 30–52 in rat GAP43) containing the phosphorylation site (Ser-41) as a red box, and the farnesylation signal, which directs lipidation of PAGFP-F, as a blue box. Right, calculated relative molecular weights. (B) Immunoblots of lysates of transfected PC12 cells expressing the respective constructs. Blots were developed using anti-GFP (left and right) and anti-GAP43 antibody (middle). GFP-tagged constructs are indicated by arrowheads, endogenous GAP43 by an arrow. The phosphomimicking GAP43 construct is not recognized by the anti-GAP43 antibody (JP91), suggesting that mutation of Ser-41 to Asp blocks binding of the antibody. Numbers to the sides of the blots indicate molecular mass standards in kilodaltons. (C) Fluorescence micrographs of PC12 cells transfected with the respective GAP43 constructs and controls. Cells were treated with NGF for 6 d. Exogenous proteins were detected using anti-GFP antibody. Note the enrichment of the GAP43 constructs in the tip of the processes. Scale bar, 50 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Expression of PAGFP-tagged GAP43 constructs in neuronally differentiated PC12 cells. (A) Schematic representation of the PAGFP-tagged GAP43 constructs. PAGFP with a farnesylation signal (PAGFP-F) and 3×PAGFP were prepared as reference constructs. PAGFP is indicated as a green box, the IQ domain (position 30–52 in rat GAP43) containing the phosphorylation site (Ser-41) as a red box, and the farnesylation signal, which directs lipidation of PAGFP-F, as a blue box. Right, calculated relative molecular weights. (B) Immunoblots of lysates of transfected PC12 cells expressing the respective constructs. Blots were developed using anti-GFP (left and right) and anti-GAP43 antibody (middle). GFP-tagged constructs are indicated by arrowheads, endogenous GAP43 by an arrow. The phosphomimicking GAP43 construct is not recognized by the anti-GAP43 antibody (JP91), suggesting that mutation of Ser-41 to Asp blocks binding of the antibody. Numbers to the sides of the blots indicate molecular mass standards in kilodaltons. (C) Fluorescence micrographs of PC12 cells transfected with the respective GAP43 constructs and controls. Cells were treated with NGF for 6 d. Exogenous proteins were detected using anti-GFP antibody. Note the enrichment of the GAP43 constructs in the tip of the processes. Scale bar, 50 μm.
Mentions: To generate a neuronal model to allow us to determine the dynamics and distribution of wild-type and modified GAP43 in living cells, we prepared a panel of constructs in which PAGFP was fused to GAP43wt and phosphoblocking (GAP43S41A) and phosphomimicking GAP43 mutants (GAP43S41D) at Ser-41 (Figure 1A). According to Simple Modular Architecture Research Tool (SMART) analysis (Schultz et al., 1998; Letunic et al., 2012), Ser-41 is located in an IQ domain, which serves as a binding site for EF-hand proteins such as calmodulin (Rhoads and Friedberg, 1997). For comparison, we used a cytosolic reference protein (3×PAGFP) and a PAGFP construct with a farnesylation signal from c-Ha-Ras fused to the C-terminus (PAGFP-F). Note that PAGFP-F most likely becomes also palmitoylated, since the sequence, which is fused to PAGFP, also contains palmitoylatable cysteine residues (Aronheim et al., 1994). Bioinformatic analysis revealed two potential palmitoylation sites in addition to the farnesylation site (CSS-Palm 2.0; Ren et al., 2008). Thus PAGFP-F could serve as a reference protein for membrane attachment, which requires at least dual lipidation (Shahinian and Silvius, 1995). The constructs were expressed in PC12 cells, which provide a well-established model for differentiating neurons. The GAP43 fusion proteins separated at an apparent molecular weight of ∼80 kDa (Figure 1B). The retarded mobility compared with the calculated molecular weight (52.6 kDa) is in agreement with the fact that GAP43 alone separates at higher molecular weight than calculated (Benowitz et al., 1987). 3×PAGFP separated at an apparent molecular weight of ∼95 kDa, which makes it appropriate as a cytosolic control construct of similar size. PAGFP-F separated at 32 kDa, close to the calculated molecular mass. We did not observe major degradation, indicating that the constructs are stable within the cells. Endogenous GAP43 and the GAP43wt and GAP43S41A fusion proteins were detected by an antibody against GAP43 (Figure 1B, middle, arrow). The antibody did not detect GAP43S41D, suggesting that the phosphomimicking mutation abolished antibody reactivity.

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

Show MeSH
Related in: MedlinePlus