U UNIVERSITY OF CINCINNATI Date: I, , hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: Mimicking B crystallin phosphorylation at serine 45 and 59 in vivo A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Masters of Science in the Department of Molecular and Developmental Biology of the College of Medicine by Shawna K.B. Hottinger B.S. Marshall University, May 2004 June 2009 Committee Chair: Jeffrey Robbins, Ph.D. Abstract The small heat shock protein B crystallin (CryAB) is involved in a myriad of cellular functions including chaperone activity, apoptosis, and protein degradation. Its prevalence in cardiac myopathies and protein aggregation disorders has generated immense interest in this protein. Recent in vitro studies advocate a role for phosphorylation in the regulation and solubility of CryAB. To determine the effects of phosphorylation on cardiac CryAB’s localization, aggregation, and function in vivo, this investigation used a cardiac-specific, inducible bi-transgenic system in which CryAB serine residues 45 and 59 were mutated singly and in combination to aspartic acid to mimic a constitutively phosphorylated state (CryABS45D, CryABS59D, CryABS45/59D). The data suggest that mimicking phosphorylation at serine 45 is benign, that a negative charge at serine 59 causes dilated hypertrophy of the heart, and that aspartic acid substitution at serine 45 and 59 causes formation of peri-nuclear intracellular aggregates and cardiomyopathy. Further in vivo studies of CryAB and its phosphorylation under normal and disease conditions are required in order to advance CryAB as a therapeutic target. iii iv Mimicking B crystallin phosphorylation at serine 45 and 59 in vivo Table of Contents Title Page ii Abstract iii Table of Contents v Introduction 1-7 Rationale for Thesis Project 7-8 Significance 8 Experimental Approach 8-11 Results & Discussion 11-27 Founder Screening 11 tTA System 12 CryABS59D 12-17 CryABS45D 17-19 CryAB S45/59D 19-22 Experimental Approach Addendum 22-23 HSPB2 23-26 Conclusion 26-27 Materials & Methods 28-33 Funding 33 Figures 34-50 References 51-57 v Introduction Small heat shock proteins (sHSP) are a highly divergent family of proteins prevalent from bacteria to higher eukaryotes (Haslbeck et al., 2005). Although exhibiting great variance in sequence and mass, most family members exhibit the following commonalities: 1) a central, conserved domain of approximately 90 residues ( -crystallin domain) flanked by two variable domains (N- and C-terminal), 2) a molecular mass of 12-43kDa, 3) formation of large, dynamic oligomers, 4) stress-responsive induction, and 5) chaperone activity via binding and sequestration of denatured protein (Augusteyn, 2004; Haslbeck et al., 2005; Sun and MacRae, 2005a). Although often involved in disease prevention due to their molecular chaperone function, sHSP have received attention due to their association with a variety of pathological conditions (Sun and MacRae, 2005b). Initially studied due to its predominance in the vertebrate eye lens, B crystallin (CryAB) is the most abundant sHSP in cardiac and skeletal muscle, constituting approximately three percent of total myofibril protein (Kato et al., 1991; Lutsch and al, 1997; Lutsch et al., 1997). Low level CryAB expression is constitutive in lens epithelium, heart, kidney, skeletal muscle, lung, and brain and can be upregulated in response to certain physiological stressors such as heat shock and hypertonic stress (Bhat et al., 1999; Bhat and Nagineni, 1989; Dasgupta et al., 1992; Dubin et al., 1989; Horwitz, 1992; Iwaki et al., 1990; Iwaki et al., 1989; Klemenz et al., 1991). The CryAB gene shares an intergenic promoter region with another sHSP, myotonic dystrophy kinase binding protein (MKBP) otherwise known as HSPB2 (Iwaki et al., 1997). While intergenic promoters often indicate co-expression or expression-repression of the head-to-tail genes, 1 it has not been determined whether HSPB2 and CRYAB have overlapping regulatory elements (Iwaki et al., 1997). Accumulating data implicate CryABwildtype in a myriad of cellular functions including autokinase activity, cytoskeletal modulation, protein degradation, apoptosis, Golgi organization, DNA binding, and molecular chaperoning (Boelens et al., 2001; den Engelsman et al., 2003; Djabali et al., 1999; Fujita et al., 2004; Gangalum et al., 2004; Horwitz, 1992; Ito et al., 2002; Kamradt et al., 2001; Kantorow and Piatigorsky, 1994; Morrison et al., 2003; Pietrowski et al., 1994; Quinlan, 2002). The high conservation of the CryAB gene between species is considered an indication of stringent structural and functional restrictions (de Jong and Hendriks, 1986). This selective degree of sequence preservation can easily be seen through comparison of the human and murine amino acid sequence (Figure 1). CryAB is associated with a number of pathologic conditions. A familial study in 1998 found that the presence of a missense mutation in CryAB, an arginine to glycine substitution at residue 120 (CryABR120G), cosegregated with the neuromuscular disorder desmin-related myopathy (DRM) (Vicart et al., 1998). A transgenic mouse model with adult, heart-specific expression of murine CryABR120G recapitulates the cardiomyopathy of the human disease, indicating causality (Wang et al., 2001a). One hundred percent of these animals die of heart failure between five and seven months of age (Wang et al., 2001a). At a cellular level, these transgenics evidence decreased CryAB solubility, formation of aggresomes, presence of soluble amyloid oligomer, and cardiomyocyte toxicity (den Engelsman et al., 2005; Sanbe et al., 2004; Sanbe et al., 2005). In addition to the CryABR120G mutation, CryABwildtype has been found in inclusion bodies, intracellular accumulations of aggregated proteins, in many human disorders including 2 Alexander disease, Lewy body disease, Alcoholic liver disease, and Alzheimer’s (Head and Goldman, 2000; Iwaki et al., 1989; Kato et al., 2001; Lowe et al., 1992; Renkawek et al., 1994; Stege et al., 1999). CryAB’s presence in cardiac myopathies and protein aggregation disorders has generated immense interest in this protein. However, whether the presence of CryAB in these myopathies is a secondary effect, a protective or maladaptive response, or an underlying cause remains largely undetermined. Mechanistic models of the in vivo activity of CryABwildtype and CryABR120G will be critical in defining CryAB’s role in human disease. In spite of high gene conservation suggesting CryAB’s necessity and its promising expression profile during cardiac development leading to hypothesized functions in myogenic differentiation and cardiac morphogenesis, the CryAB/HSPB2 double knockout mouse (KO) was not embryonic lethal, showed normal Mendelian ratios in heterozygous matings, and did not display any overt cardiac malformations (Andley et al., 2001; Brady et al., 1997; Brady et al., 2001; de Jong and Hendriks, 1986; Dubin et al., 1991; Gopal-Srivastava et al., 1995; Sugiyama et al., 2000). It did, however, develop cytoplasmic inclusion bodies in the lens and showed increased intracellular levels of desmin, a known binding partner of CryABwildtype in the heart (Andley et al., 2001; Bennardini et al., 1992; Brady et al., 1997). These mice suffered late-onset postural defects due to progressive skeletal muscular dystrophy that manifested at seven weeks of age and resulted in death at approximately forty weeks (Morrison et al., 2004). Further characterization of the KO heart revealed that it exhibited no developmental, morphological, or functional defects until challenged by a stress condition, such as ischemia, under which it displayed increased necrosis and apoptosis (Morrison et al., 3 2004). Investigators have speculated that the abundance of other cardiac sHSPs and their structural similarities to CryABwildtype and HSPB2 result in functional redundancy under unstressed conditions, but that the loss of CryABwildtype cannot be compensated for during stress (Morrison et al., 2004). Basal characterization and ischemia-reperfusion studies of the KO crossed with a transgenic CryABwildtype line (KOxCryABwildtype) suggest that CryABwildtype and HSPB2 have the non-redundant roles of preserving cardiac mechanical structure and energetic balance respectively (Pinz et al., 2008). Research concerning the importance of CryABwildtype phosphorylation is beginning to surface (Gaestel, 2002; Inaguma et al., 2001; Ito et al., 2001; Ito et al., 1997; Kamradt et al., 2002; Kato et al., 2001; Kato et al., 2002; Kato et al., 1998). CryABwildtype can be phosphorylated at serines 19, 45, and 59, depending on cell cycle state and signals from the extracellular environment (Ito et al., 1997; Kato et al., 1998). Serines 19, 45 and 59 can all be phosphorylated by 42/44 MAPK and p38 MAPK; however, serine 45 and 59 show preferential phosphorylation by 42/44 MAPK and p38 MAPK respectively (Ito et al., 1997; Kato et al., 1998). Although basal levels of phosphorylation are low, CryABwildtype phosphorylation increases during a stress response (Eaton et al., 2001; Golenhofen et al., 1998). Serines 19, 45, and 59