Protein Citation
aPKCι/λ Puls et al., 1997; Avila et al., 2002 MEK5 Lamark et al., 2003; Noda et al., 2003 NBR1 Lamark et al., 2003
PAR-4 Chang et al., 2002 p38 MAPK Sudo et al., 2000 p120 ras-GAP Ellis et al., 1990
TrkA Geetha and Wooten, 2003 RIP Sanz et al., 1999
TRAF6 Sanz et al., 2000 p56lck Park et al., 1995 Grb14 Cariou et al., 2002 Kvβ2 Gong et al., 1999 GABAC Croci et al., 2003 COUP-TFII Marcus et al., 1996 ubiquitin Vladlamundi et al., 1996
p62 Lamark et al., 2003; Wilson et al., 2003
Table 1.1: Interaction partners of p62. Abbreviation of the proteins are given in the following: aPKC = atypical protein kinase C, MEK5 = mitogen-activated protein kinase kinase 5, PAR-4 = prostate apoptosis response-4, MAPK = mitogen-activated protein kinase, p120 ras-GAP = p120 ras-specific GTPase-activating protein, TrkA = neurotrophic tyrosine kinase receptor, RIP = interacting protein, TRAF6 = TNF (tumor necrosis factor) receptor-associated factor 6, p56lck= lymphocyte-specific protein tyrosine kinase, Grb14
= growth factor receptor-bound protein 14, Kvβ2 = potassium channelβ2 sub-unit, GABAC = gamma-aminobutyric acid receptor C, COUP-TFII = chicken ovalbumin upstream promotor transcription factor
association with ubiquitin-protein conjugates constitutes the so-called sequesto-some, an alternative storage of cytoplasmic ubiquitinylated proteins which are not degradated by the proteasome in the first place (Shin, 1998).
1.11 Aim of the work
The enormous size of titin, extending over half of the sarcomere, and its location enables titin to interact with a variety of different muscle proteins, and hence get involved in signal transduction. This diversity in interactions together with its various functions makes titin a particularly interesting protein to study.
The immense size by its assembly of about 300 domains entail the flexible nature of this protein, which is obviously not suitable for crystallisation. In or-der to obtain structural information about titin, segments encompassing single or several domains are studied.
In this study, the region of titin in proximity to titin kinase as well as a connected signalling pathway are considered. Several domains of titin, namely
18 Introduction
the Ig-like domains A168 and A169 of the A-band region, M1 in the M-line and extended constructs of titin kinase are investigated. The work on titin connected pathways focuses on the proteins NBR1 and p62 and their interaction via the lately identified PB1 domain. Both proteins are a substrate of titin kinase and are linked to titin. Throughout all of the work, a special emphasis is placed on interactions of the studied domains.
Since the era of characterisation of the molecular and biochemical properties of signalling pathways in titin has just started, this work intends to shed some light on the molecular structure of domains and their interactions involved in the titin kinase downstream signalling pathway.
Chapter 2
The A-band immunoglobulin domains A168 and A169
2.1 Introduction
The assembly of large proteins from small building blocks or modules is con-sidered a common practice for generating a diversity of multifunctional pro-teins (Spitzfaden et al., 1997; Bork et al., 1996). These mosaic propro-teins are primarily extracellular proteins which excel in the immune system in recog-nition and in cell-cell interactions. But, also intracellular proteins exhibit the modular arrangement of contiguous domains such as for structural organisation of the muscle. Typically, modules of theβ-fold emerge – often with their N-and C-terminus at opposite ends – which provide the basis for easy array assembly and spacer formation. The most abundant protein module is the immunoglob-ulin (Ig) domain (Doolittle and Bork, 1993) with a large functional, structural and sequential diversity (Bork et al., 1994).
2.1.1 Immunoglobulin domains
Antibodies (immunoglobulins) were eponymous for the immunoglobulin do-mains, first discovered in the immune system. Due to their wide distribution and great number, proteins containing Ig domains cover diverse biological func-tions, such as recognition, growth, development, signalling, and carbohydrate recognition (Srinivasan and Roeske, 2005).
Ig domains display no enzymatic activity but are good in being recog-nised (Barclay, 2003). The stable fold that is resistant to proteolysis, has the ability to interact by formation of homo- and heterodimers either along theβ -strands or through loops. Their broad range of high affinity interaction resides mainly in the hypervariable loops, which are located at one end of the ellipsoid domain (Barclay, 2003).
The fold topology of Ig-like domains follows the Greek key fold with two twisted β-sheets comprising seven to nine antiparallel β-strands which form a β-sandwich structure (Richardson, 1981; Lesk and Chothia, 1982). Despite the
19
20 The A-band immunoglobulin domains A168 and A169
conserved common fold, the Ig-like domains represent distant sequence similar-ity. Similar residues within the module of about 100 residues were detected and a conserved disulphide-bridge has long been considered as the hallmark of the Ig-like domains (Williams and Barclay, 1988). Typically, it is formed in the extracellular Ig domains connecting two cysteines between strand B and F. However, many intracellular and also some extracellular Ig-like domains are devoid of disulphide bridges. If the disulphide bridge is present, it packs against an invariant tryptophan in the hydrophobic core forming the so-called ’pin’ mo-tif (Lesk and Chothia, 1982). Overall, the loops intervening the strands show variability in length and particularly the B-C and F-G loops play a key role in protein recognition as well as in determining the size of the domain.
Ig domains of giant proteins such as titin have been grouped to the I-set class (Harpaz and Chothia, 1994). The first structure of a muscle Ig-like domain, termed telokin, was solved from theC. eleganstwitchin protein (Holden et al., 1992) and became the representative of this group.
2.1.2 Interaction of titin A168-A169 and MURF
The muscle-specific RING protein MURF interacts with diverse proteins in-and outside the nucleus. One of the binding partners is the titin tin-andem Ig domain A168-A169. The interaction has been demonstrated for MURF-1 using yeast two-hybrid assays and pulldown experiments of in vitro translated pro-tein (Centner et al., 2001). Investigations regarding the binding capability of the MURF family members MURF-2 and MURF-3 to titin A168-A169 have been inconsistent so far (Centner et al., 2001; Witt et al., 2005; Pizon et al., 2002). As MURF-1 was found to bind to A168-A169, a potential role in mod-ulating the activity of titin kinase was suggested due to the close proximity of titin kinase and A168-A169 (Centner et al., 2001). Several regions of MURF-1 were mapped to contribute in binding to titin A168-A169 (Witt et al., 2005).
MURF-1 plays an important physiological role in regulating muscle degrada-tion (Bodine et al., 2001). Under atrophic condidegrada-tions in muscle, upreguladegrada-tion of MURF-1 and muscle atrophy F-box protein MAFbx was detected. Fur-thermore, MURF-1 knock-out mice were described to be resistant to muscle wasting (Bodine et al., 2001).
MURF-1 has E3-like ubiquitin ligase activity and may thereby be involved in sumoylation (Dai and Liew, 2001; McElhinny et al., 2002) or ubiquitination processes involved in proteasome-dependent proteolysis of muscle proteins (Bo-dine et al., 2001; Kedar et al., 2004; Witt et al., 2005).
Location of MURF-1 in the nucleus, where it acts in transcription regula-tion (Dai and Liew, 2001; McElhinny et al., 2002), and its interacregula-tion with titin (Centner et al., 2001) and other sarcomeric proteins (Witt et al., 2005;
Kedar et al., 2004) suggest a dynamic role as adaptor, linking myofibrillar sig-nalling with transcription control.
To structurally elucidate the basis for the interaction of the titin A168-A169 and MURF-1, the structure of A168-A169 has been solved. Since some studies