Biochemistry zyxwvut 1995,34, zyxwvu 8123-8129 8123 Interaction between Casein Kinase I1 and the 90-kDa Stress Protein, HSP90' Yoshihiko Miyata and Ichiro Yahara" Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo zyxw 113, Japan Received November 29, 1994; Revised Manuscript Received March 23, 1995@ ABSTRACT: Purified casein kinase zyxwvut I1 (CKII) aggregates and loses activity under physiological salt conditions and within the range of physiological temperatures. In accord with our previous report [Miyata, Y., zy & Yahara, I. (1992) zyxwvutsrqp J. Biol. Chem. 267, 7042-70471, we report here that HSP90 protects CKII from the aggregation and inactivation by forming soluble CKII-HSP90 complexes. Surface plasmon resonance (SPR) measurements revealed that CKII binds to immobilized HSP90 within minutes. The zyx KD of the binding is approximately lo-' M. ATP does not influence the interaction. The membrane-overlay method revealed that HSP90 binds to the catalytic CKII a subunit. Heparin, which binds to CKII a, inhibited the binding of CKII to HSP90-Sepharose. In addition, HSP9O competed with DNA for binding to CKII. Finally, SPR experiments showed that a peptide corresponding to the heparin and DNA binding site of CKII a binds to immobilized HSP90. These results indicate that HSP90, DNA, and heparin compete with each other for binding to a common site of CKII a. If the binding of CKII to DNA is biologically significant, it could be possibly regulated also by HSP90. Exposure of living organisms to environmental stresses such as elevated temperatures induces a small set of proteins called stress proteins or heat shock proteins (HSPs)' [for a review, see Lindquist and Craig (1988) and Welch (1992)l. The 90-kDa stress protein, HSP90, is ubiquitously distributed in all living organisms from bacteria to human. Like other stress proteins, the amino acid sequence of HSP90 is highly conserved among these species, suggesting that HSP90 is involved in basic processes essential for cells (Lindquist & Craig, 1988; Hickey et al., 1989). In fact, HSP90 has been shown essential for the budding yeast, Saccharomyces cerevisiae, to be viable (Borkovich et al., 1989). HSP90 is abundantly expressed in mammalian cells under normal conditions while the content of HSP90 further increases severalfold or more when cells are exposed to stresses. HSP90 has been shown to interact with various functionally key proteins. They include steroid hormone receptors (Joab et al., 1984; Catelli et al., 1985; Sanchez et al., 1985), viral oncogene products bearing tyrosine-specific protein kinase activities such as pp60"-"'" (Opperman et al., 1981; Brugge et al., 1981; Schuh et al., 1985), certain serinetthreonine- specific protein kinases (Rose et al., 1987; Miyata & Yahara, 1992; Matts et al., 1992; Stancato et al., 1993), and cytoskeletal proteins (Koyasu et al., 1986; Nishida et al., 1986; Miyata & Yahara, 1991; Minami et al., 1993). In addition, HSP90 associates with other stress proteins includ- ing HSP70 and HSP56 (FK506 binding protein, FKBP52) (Renoir et al., 1990; Sanchez et al., 1990; Perdew & ' This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan, by the Nissan Science Foundation, and by a grant from the Human Frontier Science Program. * Address correspondence to this author at 3-1 8-22, Hon-Komagome, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-5685-2264. Fax: 81-3- 5685-2932. E-mail: yahara@rinshoken.or.jp. @ Abstract published in Advance ACS Abstracts, June 1, 1995. ' Abbreviations: HSP(s), heat shock protein(s); CKII, casein kinase 11; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, poly- acrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue R-250; PVDF, poly(viny1idene difluoride); FKBP, FK506 binding protein: Tris, tris(hydroxymethy1)aminomethane: SPR, surface plasmon resonance. 0006-2960/95/0434-8 123$09.00/0 Whitelaw, 1991; Nadeau et al., 1993). HSP90 also binds ATP and Ca2+ (Csermely & Kahn, 1991; Kang & Welch, 1991). Evidence for biological significance of the interaction between HSP90 and its various target proteins has been suggested for some cases. For instance, it has been elegantly demonstrated that HSP90 is necessary for steroid-dependent transcription directed by the steroid hormone receptors (Picard et al., 1990). In addition, association of certain steroid hormone receptors with HSP90 increases affinities to their ligands by 100 times or more as compared to the free receptors (Bresnick et al., 1989; Nemoto et al., 1990). We have previously reported that casein kinase I1 (CKII) exists as complexes with HSP90 in cell lysates and that the complexes were reconstituted in vitro (Miyata & Yahara, 1992). The interaction of CKII with HSP9O has been observed also by other investigators [Csermely & Kahn, 1991; Nadeau et al., 1993; see also Shi et al. (1994)l. CKII tends to aggregate in vitro at low salt concentrations and to lose its kinase activity (Glover, 1986). It is thus possible that HSP90 might assist CKII in folding into the native conformation. This function of HSP9O is at least in part similar to that of molecular chaperones such as HSP60/ GroEL although HSP9O exerts its function through forming complexes with the target proteins [for a review, see Welch (1992) and Hendrick and Hart1 (1993); see also Wiech et al. (1992)l. CKII is known to phosphorylate a number of nuclear and cytoplasmic proteins [for a review, see Tuazon and Traugh (1990) and Issinger (1993)l. Growth factors such as epidermal growth factors stimulate severalfold the activity of CKII recovered in cell lysates [for a review, see Krebs et al. (1988), Pinna (1990), and Issinger (1993)l. Conversely, when the activity of CKII was inhibited by microinjecting anti-CKII antibodies (Lorentz et al., 1993) or when the expression of the kinase was reduced by the antisense RNA method (Pepperkok et al., 1991), cell growth was inhibited. Recently, it is suggested that CKII a binds DNA (Filhol et al., 1990), and thereby controls the expression of certain 1995 American Chemical Society