| Ankyrin is an evolutionarily conserved phosphoprotein that functions in membrane-cytoskeletal linkage in a variety of eukaryotic cells. Ankyrin function appears to be critical for membrane stability, protein localization and defining membrane domains in cells. In red blood cells, ankyrin binds AE1 anion exchanger proteins in vitro. However, the stoichiometry and kinetics of this interaction is very complicated. The functional significance of this in vitro complexity, as well as the relevant parameters for extrapolating these data towards understanding the in vivo interactions are not known. We investigated assembly, trafficking and cytoskeletal interactions of AE1-ankyrin complexes in chicken erythroid cells. Our results reveal that AE1 cytoskeletal assembly is dependent on plasma membrane to Golgi recycling of this plasma membrane polypeptide. Ankyrin associates with AE1 in detergent-soluble complexes, immediately after synthesis, suggesting that ankyrin may function in trafficking of the membrane anion transporter. Conversely, the detergent-resistance properties of only a small fraction of cellular AE1 are actually dependent on cytoskeletal ankyrin. AE1 insolubility appears to be dependent on recruitment of this membrane protein into Triton X-100 insensitive lipid microdomains. Additionally, ankyrin-AE1 association is very dynamic, involving assembly, disassembly and turn over of ankyrin from AE1-containing complexes. Ankyrin cytoskeletal properties are regulated by phosphorylation level of this membrane-cytoskeletal adapter. Our studies are the first of its kind to demonstrate in vivo consequences of ankyrin phosphorylation. The level of ankyrin phosphorylation is regulated by a concerted action of the ankyrin kinase, casein kinase II, and a serine-threonine phosphatase. Moreover, casein kinase II (CK2) is constitutively associated with ankyrin thus providing a mechanism for recruitment of this kinase to membrane-cytoskeletal junction sites. The presence of ankyrin-CK2 complexes suggest that ankyrin may be involved in rapid modulation of membrane-cytoskeletal interactions in response to various signal transduction events. |
