Abstract
We recently cloned a cDNA encoding the 29-kDa subunit of human red blood cell regulator (REG), a potent activator of the multicatalytic protease (Realini, C., Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1994) J. Biol. Chem. 269, 20727-20732). The sequence of this subunit contains 28 "alternating" lysine and glutamic acid residues (a KEKE motif). Similar regions are present in a number of Ca(2+)-binding proteins, and using standard filter assays, the recombinant protein is shown to bind 45Ca2+ and ruthenium red. 45Ca2+ is also bound to a ubiquitin extension protein containing the 28-residue KEKE region from the 29-kDa REG subunit. Thus, the 29-kDa REG subunit is a Ca(2+)-binding protein, and its KEKE region is able to bind divalent cations. Ca2+ reversibly inhibits the enhanced peptidase activity of complexes between the multicatalytic protease and recombinant REG. This raises the possibility that multicatalytic protease activity is regulated by calcium in vivo.
Highlights
The multicatalytic protease (MCP)1 or 20 S proteasome is a large (ϳ700 kDa) multimeric enzyme found in eukaryotes, prokaryotes, and archaebacteria
We have recently cloned and expressed the gene for the 29-kDa subunit of human regulator, and we have shown that the recombinant species activates MCP in a manner very similar to the molecule purified from human red blood cells [19]
Because KEKE motifs are present in Ca2ϩ-binding proteins and because Ca2ϩ is an important regulator of cellular processes [23, 24], we asked whether the 29-kDa REG subunit is capable of binding Ca2ϩ
Summary
(Received for publication, September 12, 1995, and in revised form, October 20, 1995). Ca2؉ reversibly inhibits the enhanced peptidase activity of complexes between the multicatalytic protease and recombinant REG. In an ATP-dependent reaction, MCP can associate with a regulatory complex that contains at least 15 subunits with apparent molecular masses between 25 and 110 kDa [11,12,13] This generates the ATP-dependent 26 S protease. KEKE motifs are found in a variety of proteins including subunits of the 26 S protease and MCP, microtubule-associated protein 1B, myosin phosphatase, triadin, and chaperonins such as hsp and hsp90 [20] They are present in the calcium-binding proteins calreticulin, calnexin, endoplasmin, and Ca2ϩ-dependent adenosine triphosphatase (Ca2ϩ-ATPase). Concentrations of Ca2ϩ in the midmicromolar range reversibly inhibit the peptidase activity of rREG29K-MCP complexes
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