Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.
Bottom Line: How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known.A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner.Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.Show MeSH
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Mentions: To further substantiate the significance of the formation of the complex for sorting and TGN Ca2+ entry, we aimed at elucidating the consequence of impaired CFL-1 and actin binding to SPCA1. As described previously, SPCA1 binds to active CFL-1, and this binding recruits CFL-1 to the TGN (von Blume et al., 2011). We have also reported previously that inactivation of CFL-1 by overexpression of LIMK, which phosphorylates CFL-1, or knockdown of slingshot, which dephosphorylates CFL-1, interferes with protein secretion (von Blume et al., 2009). To test if an inactive CFL-1 phosphomimetic S3E mutant that does not bind to SPCA1 interferes with Ca2+-dependent protein sorting, HeLa cells were transfected with control or ADF/CFL-1 siRNAs as well as a siRNA-resistant HA-tagged rat CFL-1 (rCFL-1) or rCFL-1-S3E, respectively. To test the knockdown and the expression of CFL-1 and the siRNA-resistant mutants, lysates were analyzed by Western blotting with CFL-1 and ADF antibodies, respectively. ADF/CFL-1 was successfully depleted by siRNAs, and overexpression of HA-rCFL-1 wild type (wt) and S3E in ADF/CFL-1–depleted cells occurred at similar levels (Fig. 7 A).
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.