Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.
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.
Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.Show MeSH
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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.
Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.