Research Interests: Protein Phosphatases ( Molecular mechanisms of carcinogenesis; Mechanisms of cardioprotective agents in ischemic myocytes). The reversible phosphorylation of many proteins determines their biological activity and is a key mechanism controlling intracellular events as diverse as metabolism, contractility, cell division, hormonal action and signal transduction. This phosphorylation/dephosphorylation of proteins occurs on specific serine, threonine, or tyrosine residues and represents a dynamic equilibrium between the activities of both protein kinases and protein phosphatases. In my laboratory, we are currently studying the molecular mechanisms associated with cancer, coronary heart disease and diabetes. The common theme of our research is the functions of specific protein phosphatases. A major goal of our cancer research is to identify and clone novel protein phosphatases that may play a role in the aberrant proliferative behavior of neoplastic cells. We have also identified a number of natural marine toxins with specific and potent inhibitory activity against certain protein phosphatases. Some of these "toxins" alter the cell cycle progression of tumor cells in culture, and we are interested in determining if these compounds have the potential for development into novel drugs for the treatment of certain cancers. We are also developing techniques to better use these phosphatase inhibitors for the identification of novel protein phosphatases and to study the roles of specific phosphatases in normal and aberrant cellular functions. We are also interested in the identification and development of novel type specific inhibitors. To See Current Projects in the Laboratory: The focus of my research is to elucidate the roles of human serine/threonine protein phosphatases (PPases) in the regulation of signal transduction cascades associated with cellular responses to stress and agents that influence cellular proliferation/differentiation. Currently, this effort is funded by two R01-grants from NIH, and sponsored research agreements from pharmaceutical companies. The goal of one project is to further test the hypothesis that protein phosphatases play an important role in cell cycle progression. Therefore, the interference of the "normal" activity of certain PPases may contribute to the aberrant proliferative behavior of neoplastic cells. This project began with the identification, purification and characterization of a novel PPase from bovine brain. Subsequent studies have lead to the identification, cloning and characterization of a structurally related human PPase and the human gene that encodes another. To determine the roles of these newly identified human PPases in the regulation of cell cycle progression, we have devoted a considerable effort to the identification and characterization of novel PPase inhibitors. These studies have proven very successful, revealing that the toxic properties of a large family of cyanobacterial produced cyclic peptides (see study 1, study 2, study 3, study 4) originates from their extremely potent and relatively non-selective inhibitory activity against the PP1/PP2A family of PPases. In addition, our studies suggest that the antitumor activity of fostriecin, a proprietary compound currently under evaluation in clinical trials, results from the selective inhibition of either an individual human PPase, or a subset of related PPases. (see study 1, study 2) In an effort to better understand the mechanism(s) underlying the antitumor activity of fostriecin, we have been comparing the cellular effects produced by fostriecin treatment with those produced by other semi-selective PPase inhibitors. To the formation of date, these studies indicate that the inhibition of a fostriecin sensitive PPase(s) interferes with normal mitotic spindles. This results in the formation of multiple aberrant mitotic spindles, leading to G2/M-phase growth arrest and then the onset of an apoptotic response. (see study 1, study 2) We are currently conducting studies designed to determine which PPases are critical for mitotic spindle formation and the molecular mechanisms by which the inhibition of fostriecin sensitive PPase induces G2/M-phase growth arrest. Although the antitumor activity of fostriecin appears promising, toxicity may limit the clinical use of fostriecin as an antitumor drug in humans. To identify less toxic (i.e. more specific) PPase inhibitors we have entered into an industrial collaboration. The goal of this project is to identify and characterize antisense oligonucleotides that specifically inhibit the expression of the known human PPases. Through the identification of truly specific inhibitors we hope to identify compounds that are less toxic yet retain the ability to inhibit tumor cell growth. The long-term goal of this project is to obtain compounds that specifically inhibit the expression of all known human PPases and explore their potential pharmacological uses in man. To date, we have been able to develop chimeric second generation antisense oligonucleotides that potently (IC50<50 nM) inhibit the expression of PP1 (isoform 1) and PP5, providing us with the unique ability to "knock out" their expression in human cells. Our studies indicate that neither PP5 or PP1 1 has an apparent effect on mitotic spindle formation. However, the inhibition of their expression has produced some very interesting findings. Our studies indicate that PP5 acts as a suppressor of a G1/S-phase checkpoint control mechanism utilized by both glucocorticoid receptors and the p53 tumor suppressor protein. (see study 1, study 2) The data suggest that PP5 is acting via the cyclin dependent kinase inhibitor protein, p21, and that PP5 functions as a suppressor of both hormone- and DNA damage /stress-induced signaling networks which function to suppress G1/S-phase cell progression. Understanding the relationship of PP5 in DNA damage induced and glucocorticoid induced responses associated with checkpoint control mechanisms regulating G1 and G2/M-phase growth arrest is an area of active research in the lab. Our other major research project is designed to determine how the inhibition of specific protein phosphatases provides protection to ischemic cardiac tissue. These studies originate from the observation that in addition to its ability to inhibit tumor cell growth, fostriecin has a pronounced cardioprotective effect in ischemic rabbit hearts. (see study 1, study 2, study 3) At concentrations that have no apparent toxicity, we have recently demonstrated that fostriecin reduces the size of a myocardial infarction induced by a controlled coronary occlusion from ~33% to ~8% of the risk zone. At the molecular level we have shown that fostriecin inhibits the activity of serine/threonine protein phosphatases types 1 (PP1), and 2A (PP2A), suggesting that the cardioprotective properties of fostriecin arise from its inhibitory activity against these or a related PPase. However, since fostriecin provides protection at a concentration that has no effect on PP1 and no apparent difference in PP1 activity was noted in control and ischemic preconditioning rabbit hearts, the inhibition of PP1 activity is not likely to underlie the protective response produced by fostriecin. The goal of this proposal is to test the hypothesis that the inhibition of certain serine/threonine protein phosphatases (PPases) is the biochemical basis for the cardioprotective properties of fostriecin observed in ischemic heart tissue. Through a better understanding of the molecular events by which the inhibition of these enzymes limits myocardial tissue damage, we hope to gain insight into the mechanisms that produce protection. Such insight should aid in the development of new and improved methods for the medical management of an acute myocardial infarction. In addition to using the toxins to study phosphatases, we are also using phosphatases to detect toxins. Okadaic acid is the principal toxin associated with diarrhetic shellfish poisoning (DSP), which is a serious and globally widespread seafood related illness. Since okadaic acid is a very potent inhibitor of protein phosphatase type 2A (PP2A), we are interested in developing an enzyme based assay for the detection of DSP-toxins in shellfish. Such a test should be useful for the protection of human health and should benefit the fishing industry by providing a tool to aid in the efficient management of potentially toxic seafood. Understanding the etiology of diseases induced by toxic protein phosphatase inhibitors, such as DSP in man and "net-pen liver disease" in salmon, is also a peripheral interest of my lab. Representative Publications: Cheng A, Dean N, and Honkanen RE. Serine/threonine protein phosphatase type 1 1 (PP1 1) is required for the completion of cytokinesis in human A549 lung carcinoma cells. J. Biol. Chem. In press, 2000. Connor JH, Kleeman T, Barik S, Honkanen RE, and Shenolikar S. Importance of the ß12-ß13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors. J. Biol. Chem. 1999; 274(32):22366-22372. Zuo Z, Urban G, Scammell JG, Dean NM, McLean TK, Argon I, and Honkanen RE. Ser/thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest. Biochemistry, 1999; 38 (28):8849-8857. Huang X, Swingel MR, Honkanen RE. Photoreceptors serine/threonine protein phosphatase type 7 (PP7): cloning, expression and functional analysis. In: Methods in Enzymology Vertebrate Phototransduction and the visual cycle, Volume 1 (K. Palczewski ed) In press, 1999. Wang L, Bhattacharjee A, Zuo Z, Hu F, Honkanen RE, Berggren PO, Li M. A low voltage-activated Ca2+ current mediates cytokine-induced pancreatic beta-cell death. Endocrinology 1999; 140(3):1200-1204. Cheng A, Balczon R, Zuo Z, Koons J, Walsh AH, and Honkanen RE. Fostriecin-mediated G2-M-phase growth arrest correlates with abnormal centrosome replication, the formation of aberrant mitotic spindles, and the inhibition of serine/threonine protein phosphatase activity. Cancer Research. 1998; 58(16):3611-9. Weinbrenner C, Baines CP, Lui G-S, Armstrong SC., Ganote CE, Walsh AH, Honkanen RE, Cohen MV, and Downey JM. Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia. Circulation: 1998; 98(9):899-905. Zuo Z, Dean NM, Honkanen RE. Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21WAF1/Cip1 and mediates growth arrest. J. Biol. Chem. May 15, 1998; 273(20) 12250-12258. Huang X, and Honkanen RE. Molecular cloning, expression and characterization of a novel human serine/threonine protein phosphatase, PP7, that is homologous to Drosophila retinal degeneration C gene product (rdgC). J. Biol. Chem. 1998; 273(3):1462-1468. Larsson O, Barker CJ, Sjöholm Å, Carlqvist H, Michell RH, Bertorello A, Nilsson T, Honkanen RE, Mayr GW, Zwiller J, Berggren P. Inhibition of phosphatases and increased Ca2+channel activity by inositol hexakisphosphate Science October 17, 1997; 278(5337):471-474. Walsh AH, Cheng A, and Honkanen RE. Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A. FEBS Letts.1997; 416:230-234. Diwan AH, Honkanen RE, Schaeffer RC Jr, Strada SJ, and Thompson WT. Inhibition of serine-threonine protein phosphatases decreases barrier function of rat pulmonary microvascular endothelial cells J. Cell Physiol. 1997; 171(3):259-270. Armstrong SC, Kao R, Shivell LC, Downey JM, Honkanen RE, and Ganote CE. Comparison to in vitro preconditioning response of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. J. Mol. Cell. Cardiol. 1997; 29:3009-3024. Huang X, Cheng A, and Honkanen RE. Genomic organization of the human PP4 gene encoding a serine/threonine protein phosphatase (PP4) suggest a common ancestry with PP2A. Genomics 1997; 44:336-343. Honkanen RE, Mowdy DE, and Dickey RW. Detection of DSP-toxins, okadaic acid, and dinophysis toxin-1 in shellfish by serine/threonine protein phosphatase assay. Journal of AOAC (Association of Official Analytical Chemists) International 1996; 79(6):1336-1343. Honkanen RE, Stapleton JD, Bryan DE, and Abercrombie J. Development of a protein phosphatase based assay for the detection of phosphatase inhibitors in crude whole cell/animal extracts Toxicon 1996; 34(11/12):1385-1392. Sjöholm Å, Honkanen RE, and Berggren PO. Inhibition of serine/threonine protein phosphatases by secretagogues in insulin-secreting cells. Endrocrinology 1995; 136(8):3391-3397. Honkanen RE, Caplan FR, Baker KK, Baldwin CL, Bobzin SC, Bolis CM, Cabrera GM, Johnson LA, Jung JH, Larsen LK, Levine IA, Moore RE, Nelson CS, Patterson GML, Tschappat KD, Tuang GD, Boynton AL, Arment AR, An J, Carmichael WW, Rodland KD, Magun BE, and Lewin RA. Protein phosphatase inhibitory activity in extracts of cultured blue-green algae (Cyanophyta). J. Phycol. 1995; 31:478-486. Critz SD, and Honkanen RE. Protein phosphatases and their role in the regulation of membrane currents in sensory neurons of Aplysia. Neuroprotocols: a companion to Meth. in Neruobiol. 1995; 6(1):78-83. Ämmälä C, Eliasson L, Bokvist K, Berggren PO, Honkanen RE, Sjöholm Å, and Rorsman P. Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic cells. Proc. Natl. Acad. Sci. 1994; 91:4343-4347. Honkanen RE, and Boynton AL. Serine/threonine protein phosphatases and their inhibitors. In: Protein Kinase C. (J. F. Kuo, ed), Oxford University Press. pp 305-324, 1994. Honkanen RE, Codispoti BA, Tse K, and Boynton AL. Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases. Toxicon. 1993; 32(3):339-350. Sjöholm Å, Honkanen RE, and Berggren PO. Characterization of Serine/threonine protein phosphatases in RINm5F insulinoma cells. Bioscience Reports. 1993; 13(6):349-358. Prinsep MP, Caplan FR, Moore RE, Patterson GML, Honkanen RE and Boynton AL. Microcystin-LA from a Blue-green Algae belonging to the Stigonematales. Phytochemistry 1992; 31(4):1247-1248. Honkanen RE, Dukelow M, Moore RE, Zwiller J, Khatra BS and Boynton AL. Cyanobacterial nodularin is a potent inhibitor of Type 1 and Type 2A protein phosphatases. Mol. Pharmacol. 1991; 40:577-583. Honkanen RE, Zwiller J, Daily SL, Khatra BS, Dukelow M, and Boynton AL. Identification, purification and characterization of a novel serine/threonine protein phosphatase from bovine brain. J. Biol. Chem. 1991; 266(10):6614-6619. Honkanen RE, Zwiller J, Moore RE, Daily SL, Khatra BS, Dukelow M, and Boynton AL. Characterization of microcystin-LR: A potent inhibitor of Type 1 and Type 2A protein phosphatases. J. Biol. Chem. 1990;265(32):19401-19404. Mordan LJ, Dean NM, Honkanen RE and Boynton AL. Okadaic acid: a reversible inhibitor of neoplastic transformation of mouse fibroblasts. Cancer Commun. 1990; 2:237-241.
 
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