a b s t r a c tApoptosis, an essential and basic biological phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a number of the mechanisms by which a protein's contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique amongst the Bcl-2 family, in that it is rich in experimentally confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage and phosphorylation, which influence the protein's stability, localisation, dimerization and function. Here we review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex molecular control of Mcl-1 with cellular state.

Cytoplasmic dynein 1 is a minus-end-directed microtubule motor that is required for a wide range of activities involving movement or anchoring of cellular structures (Vallee et al., 2004). These activities include separation of chromosomes during mitosis, cell migration, and the movement and localisation of substrates such as mRNAs, signalling complexes and membrane organelles. To understand how dynein function over such a diverse range of activities is regulated, much attention has focussed on the composition of the dynein motor complex and the roles of accessory proteins.Dynein can be purified as a 1.6 MDa complex. This contains two copies of a motor subunit (dynein heavy chain; DHC), each of which comprises an AAA ATPase region involved in force generation and a stem region that connects the ATPase to the remaining, regulatory and cargo-binding subunits of the dynein complex. These subunits include dynein intermediate chain (DIC), dynein light intermediate chain (DLIC), and dynein light chains (DLCs) (Pfister et al., 2006). Dynein engages several accessory proteins or protein complexes, which might regulate its motor and/or cargo-binding activities. The best characterised of these is dynactin, a multiprotein complex that is almost universally associated with dynein-dependent functions (Schroer, 2004). More recently, several additional dynein-interacting proteins have been identified using a screen for Aspergillus nidulans mutants that are defective for nuclear distribution. These include NudF (Xiang et al., 1995) and NudE (Efimov and Morris, 2000), the higher eukaryote counterparts of which are Lissencephaly1 (LIS1) and NDE1, respectively. An NDE1-related protein, NDEL1 (also referred to as NudEL) has also been identified Sasaki et al., 2000). LIS1 interacts directly with DHC via sites on the first AAA domain and the stem region (Sasaki et al., 2000;Tai et al., 2002), with DIC (Tai et al., 2002), and with the p50 subunit of dynactin (Tai et al., 2002). NDEL1 and NDE1 also bind to dynein. However, the mode of binding might not be conserved here, because NDEL1 has been reported to bind HC (Sasaki et al., 2000), whereas NDE1 binds to DIC and the LC8 isoform of DLC (Stehman et al., 2007). In addition, NDE1 (Efimov and Morris, 2000;Feng et al., 2000) and NDEL1 (Derewenda et al., 2007;Niethammer et al., 2000;Sasaki et al., 2000) bind to LIS1 directly, and to each other (Bradshaw et al., 2009). There are currently two models to explain how these effectors modulate dynein function. They might regulate dynein motor activity directly (Mesngon et al., 2006;Yamada et al., 2008), or they might have a role in targeting dynein to cargoes (Liang et al., 2007;Vergnolle and Taylor, 2007).LIS1, NDE1 and NDEL1 have been implicated in many dyneinmediated activities, including cell migration, (Ding et al., 2009;Dujardin et al., 2003;Feng and Walsh, 2004;Kholmanskikh et al., 2003;Sasaki et al., 2005;Shu et al., 2004;Tanaka et al., 2004;Tsai et al., 2007;Tsai et al., 2005) , 2008). Mutations in or haplo-insufficiency of mammalian...

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1β instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (Mφ) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mφ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro Mφ activation, determined as release of the CXC chemokine KC and IL-1β, was inhibited by the more selective MC3-R agonist γ2-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1β release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of γ2-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal Mφ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

In this study we have characterized the anti-inflammatory profile of a selective melanocortin type 3 receptor (MC3-R) ligand [D-Trp8]-gamma-MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, [D-Trp8]-gamma-MSH activated MC3-R (as tested by accumulation of cyclic AMP) and inhibited (approximately 50%) the release of interleukin (IL)-1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3-R null mice. In vivo, administration of 3-30 microg [D-Trp8]-gamma-MSH significantly inhibited leukocyte influx and cytokine production in a model of crystal-induced peritonitis, and these effects were absent in MC3-R null mice or blocked by coadministration of an MC3-R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (approximately 70%) by systemic or local administration of [D-Trp8]-gamma-MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3-R agonists as novel anti-inflammatory therapeutics.