First identified at the intracellular surface of the erythrocyte plasma membrane, spectrins (Sp) are now known to be the central components of the membrane skeleton, a ubiquitous and complex spectrin-actin scaffold located under the lipid bilayer of metazoan animal cells (for review, see references 4
). Numerous studies on red cells, particularly those in hereditary hemolytic anemia, have clearly established the organization of the erythrocyte skeleton and its importance in maintaining erythrocyte shape, stability, and deformability. Spectrins are giant extended flexible molecules composed of two subunits (αI and βI in red cells) which intertwine to form αβ heterodimers. Spectrin exists as elongated tetramers resulting from self-association of αβ heterodimers. Sp tetramers constitute the filaments of the lattice, the nodes of which are crossed-linked by short actin filaments. This spectrin-based skeleton is bound to various transmembrane proteins through two connecting proteins, ankyrin and protein 4.1.
In nonerythroid mammal cells, α (αI and αII) and β (βI to βV) chains are encoded by two and five genes, respectively, each of these genes producing several isoforms by alternative splicing. Despite this diversity, all Sp chains present the same structural organization mainly made up of a succession of triple-helical repeat units, 22 for α chains and 17 for β chains except βV, which has 30 repeats. These units are characteristic of spectrin family members. They are about 106 amino acids long and folded in a coiled-coil structure made up of three helices (A, B, and C). Beside these repeat units, spectrin isoforms can also contain several interacting domains, such as SH3 domain, EF hands, PH domains, and binding domains for ankyrin, actin, protein 4.1, and calmodulin.
In nonerythroid cells, spectrin isoforms are not evenly distributed at the plasma membrane. Spectrins are also present in the Golgi apparatus, in cytoplasmic vesicles (16
), and in the nucleus (31
). Several mechanisms appear to control spectrin dynamic distribution at the protein level, such as serine phosphorylation (19
) and proteolysis by calpain and caspase. Spectrin binds Ca2+
and calmodulin which regulate spectrin binding to the membrane (43
The multiple physiological functions attributed to spectrins are related to both their cellular locations and the nature of proteins that interact with them. Spectrins and the spectrin-based skeleton are considered to participate in the formation and maintenance of specialized plasma membrane domains in epithelial cells (17
), in neurons (5
), and in striated muscle cells (7
). They are considered to stabilize integral membrane proteins, to reduce their endocytic rate, and to confer resiliency and durability on the membrane itself. Recent studies also suggest that spectrin may play a role in membrane protein sorting, vesicle trafficking (3
), endocytosis (27
), and neurite outgrowth (22
). The recent description of spectrin mutations in quivering mice that manifest auditory and motor neuropathies (35
) confirms their important functions in the maintenance of specialized subcellular domains.
The involvement of spectrins in many diverse physiological processes can be explained by their modular structure that combines numerous protein-interacting domains in a number of different isoforms. One approach for obtaining insight into the function(s) of spectrins is to define the proteins interacting with its domains in specific cell types. We have focused our study on a particular domain of 350 residues located within the middle part of the αII-spectrin. This area contains two repeat units (α9 and α10) together with several additional sequences. These additional sequences include (i) an SH3 domain, (ii) a calmodulin binding site, and (iii) two cleavage sites for proteases, such as calpains and caspase 3.
SH3 domains are 60-amino-acid-long sequences that are present in many signaling and cytoskeletal proteins. Despite the modest sequence homology, the three-dimensional structure is well conserved. They mediate protein interactions by binding short proline-rich sequences bearing the consensus motif PXXP, where X is any amino acid. The αII-Sp SH3 domain is highly conserved between species, with 100% identity between birds and mammals, suggesting important and conserved functions.
The protein E3B1, a substrate for tyrosine kinase, has been identified as a ligand for the αI-Sp SH3 domain (51
) but no partner has been clearly defined for the αII-Sp SH3 domain. Using the yeast two-hybrid system, we identified isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) as a specific partner for the αII-Sp SH3 domain. As not reported previously to our knowledge, we demonstrated that αII-Sp was tyrosine phosphorylated in cells. We identified one tyrosine residue (Y1176) that is phosphorylated and dephosphorylated in vivo. This residue is located in the specific calpain cleavage site, near the SH3 domain, and is an in vitro substrate for two tyrosine kinases of the Src family, Src and Lck, and for the isoform A of LMW-PTP. Phosphorylation of this residue antagonizes calpain proteolytic activity. LMW-PTP A can dephosphorylate phosphotyrosine 1176 and so modulate spectrin susceptibility to calpain.