Abstract. We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gtll expression library. Analysis of 800 residues of deduced amino acid sequence at the amino-terminal end revealed a strikingly conserved domain of "~ 230 residues that shows a high degree of sequence similarity to the amino-terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrinrelated proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino-terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.

The complete amino acid sequence of a 55-kDa erythrocyte membrane protein was deduced from cDNA clones isolated from a human reticulocyte library. This protein, p55, is copuriflied during the isolation of dematin, an actinbundling protein of the erythrocyte membrane cytoskeleton.Fractions enriched in p55 also contain protein kinase activity that completely abolishes the actin-bundling property of purified dematin in vitro. The predicted amino acid sequence of p55 does not contain any consensus sequence corresponding to the catalytic domains of protein kinases but does contain a conserved sequence found in the noncatalytic domains ofoncogeneencoded tyrosine kinases. This conserved src homology 3 (SH-3) motif appears to suppress the tyrosine kinase activity of various oncoproteins and has also been found in several plasma membrane associated proteins involved in signal transduction. Northern blot analysis indicated that p55 mRNA was constitutively expressed during erythropoiesis and underwent 2-fold amplification after induction of K562 erythroleukemia cells toward the erythropoietic lineage. The abundant expression of p55 mRNA, along with protein 4

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.

No abstract