[CANCER RESEARCH 58. 1095-1098. March 15. 1998| Advances in Brief T-Loop Deletion of CDC2 from Breast Cancer Tissues Eliminates Binding to Cyclin Bl and Cyclin-dependent Kinase Inhibitor p2l ' Tomohiko Olita/ Kazuki Okamoto, Fumihide Isohashi, Kiyotaka Shibata, Mamoru Fukuda, Susumu Yamaguchi, and Yue Xiong First Department of Surgery IT. 0.. M. F.. S. Y.ÃOE and Department of Bioche mislry IK. O., F. I.], St. Marianna University School «/Medicine. Kawasaki 21 fi. Japan: Department of Biotechnology, School of Science and Engineering, Ishinoinaki Senshu University, Ishinonuiki 9N6-RO. Japan IK. S.¡: and Department of tìiochetnislry and Biophv\u-\. Lineberger Comprehensive Cancer Center. University of North Carolina at Chapel Hill. Chapel Hill. North Carolina 27599 [T. O., Y. X.] Abstract The eukaryotic cell cycle is regulated by a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). Monomeric free CDKs do not possess enzymatic activity, largely due to the steric hin drance caused by the T-loop at the entrance of the catalytic cleft, making ATP inaccessible to the substrate. Binding of a cyclin, primarily to the NH2-terminal lobe of the CDK that surrounds the PSTAIRE helix, in duces a large conformational change in the PSTAIRE helix of the CDK and also causes the T-loop to move out of the way of the catalytic cleft. We identified from breast cancer tissues a novel variant of human CDC2, termed CDC2AT, that lacks 171 nucleotides corresponding to 57 amino acids, which compose most of the T-loop. CDC2AT was detected in 10 of 14 breast cancer tissues analyzed, whereas it was not detectable in diploid human fibroblast cell lines or in interleukin 2-stimulated normal human lymphocytes. CDC2AT protein is unable to complex with cyclin Bl and lacks histone HI kinase activity. CDC2AT also fails to bind to the CDK inhibitor p21. These results indicate that the T-loop not only plays a key role in keeping a free CDK in its inactive state but also in facilitating CDK activation by promoting cyclin binding. Introduction In eukaryotes, the cell cycle is coordinated by several protein kinases composed of a CDK1 subunit and its corresponding regulatory cyclin subunit. CDC2. the prototypic CDK, functions in conjunction with mitotic cyclin partners to control mitosis (1). Regulation of CDC2 activity is a complex process that involves cyclin binding, subunit phosphorylations. CDK inhibitor binding, and cyclin degra dation. Like all other CDKs, free monomeric CDC2 is inactive, and binding with a cyclin is an essential step in its activation. Complete activation of CDC2 activity also requires an activating phosphoryla- tion by CDK-activating kinase on residue Thr-161. located on the T-loop (2). Conversely, binding with a CDK inhibitor or elimination of a cyclin from a CDC2-cyclin complex via degradation leads to inhibition and inactivation of CDC2 (1, 3). The structure analyses of free CDK2 and the CDK2-cyclin A complex revealed the molecular basis of CDK activation by cyclin binding (4, 5). In addition to a bilobal structure shared by other protein kinases, such as cyclic AMP-dependent kinase, free CDK2 uniquely contains an a-helical region (aL12, Gly-147 to Gly-153 in human CDK2; equivalent to Gly-149 to Gly-155 in human CDC2) that holds Received 6/27/97; accepted 1/23/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported by a Cirant-in-Aid for General Scientific Research from the Ministry of Education. Science, Sports and Culture of Japan and by a United States Public Health Sen-ice Grant CA-65572 from the NIH (to Y. X.). 2 To whom requests for reprints should be addressed, at 22-068 Lineberger Building. University of North Carolina at Chapel Hill. Chapel Hill. NC 27599. Phone: (919) 962- 2138: Fax: (919)966-8799; E-mail: tomo@imap.unc.edu. 1 The abbreviations used are: CDK, cyclin-dependent kinase: RT-PCR. reverse tran- scription-PCR; HA. hemagglutinin: CMV. cytomegalovirus; IP. immunoprecipitation. an adjacent large loop, the T-loop (Asp-145 to Ala-170 in CDK2; equivalent to Asp-147 to Glu-172 in CDC2), in a position that almost completely blocks the protein substrate binding cleft. Binding of cyclin A causes a large confonnational change in CDK2 that melts the aL12 helix and moves the T-loop away from the catalytic cleft, thereby reconfiguring CDK2 to resemble that of an active cyclic AMP-dependent kinase and allowing substrates to bind. The primary binding sites between cyclins and CDKs involve the conserved cyclin box in the cyclin subunit and the conserved PSTAIRE helix in the CDK subunit. The key role of the PSTAIRE helix as the primary cyclin-binding site is consistent with previous biochemical and ge netic data showing that mutation of the PSTAIRE domain eliminates cyclin binding and that association with a cyclin blocks the binding of anti-PSTAIRE antibody (6, 7). The cyclin box also makes contact with the NH^-terminal portion of the T-loop (residues 150-157 in human CDK2: equivalent to residues 152-159 in CDC2). Here, we identify a variant form of human CDC2 mRNA from breast cancer tissues that lacks amino acid residues 107-163, which include most of the T-loop and the aL12 helix, and the function of this variant protein was analyzed by transient transfection.4 Materials and Methods Tissue Specimens. Breast cancer tissues were obtained from 14 patients undergoing mastectomy or lumpectomy. Noncuncerous breast tissues were also obtained from two of these breast cancer patients. Thyroid cancer tissues were obtained from three patients undergoing thyroidectomy. The blocks of tissue were snap-frozen in liquid nitrogen and preserved at -80°C until use. Cell Culture and Synchronization. A human breast carcinoma cell line. MCF-7. an osteosarcoma cell line. Saos-2. and two human leukemia cell lines. HL-60 and Daudi. were obtained from American Type Culture Collection (Rockville, MD). Two normal diploid human fibroblast cell lines. HDF and NHLF. were obtained from Morinaga IBS (Yokohama. Japan) and BioWhit- taker (Walkersville. MD). respectively. An adenocarcinoma cell line Tousal was established in this laboratory (8). Cells were cultured in DMEM (mono- layer cells) or RPMI 1640 (suspension cells), supplemented with 10% FCS. 2 niM glutamine. 100 units/ml penicillin, and 0.1 nig/ml streptomycin in a humidified chamber in 5% CO, at 37°Cand harvested during exponential growth. Human lymphocytes were obtained from peripheral blood of three healthy volunteers by Ficoll-Paque density gradients method and stimulated by 200 units/ml interleukin 2 (Shionogi. Osaka. Japan), using the same medium as described above. To arrest the cells either at mitosis or at the G,-S transition of the cell cycle, exponentially growing cells were incubated for 24 h with nocodazole (Sigma Chemical Co.. St. Louis. MO) at a final concentration of 0.1 /xg/ml or hydroxyurea (Sigma) at a final concentration of K) IHM.respec tively. RT-PCR Analysis. Poly(A)* RNA was isolated from 100 mg of frozen tissue samples or 5 X IO6 culture cells using the QuickPrep micro mRNA purification kit (Pharmacia. Uppsala. Sweden) and was then converted to 4 The nueleotide sequence dala reported here have been deposited in the GenBank database (accession no. D88357). 1095