Cell signalling pathways that regulate proliferation and those that regulate programmed cell death (apoptosis) are co-ordinated. The proteins and mechanisms that mediate the integration of these pathways are not yet fully described. The phosphoprotein PEA-15 (phosphoprotein enriched in astrocytes) can regulate both the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway and the death receptor-initiated apoptosis pathway. This is the result of PEA-15 binding to the ERK/MAPK or the proapoptotic protein FADD (Fas-activated death domain protein) respectively. The mechanism by which binding of PEA-15 to these proteins is controlled has not been elucidated. PEA-15 is a phosphoprotein containing a Ser-104 phosphorylated by protein kinase C and a Ser-116 phosphorylated by CamKII (calcium/calmodulin-dependent protein kinase II) or AKT. Phosphorylation of Ser-104 is implicated in the regulation of glucose metabolism, while phosphorylation at Ser-116 is required for PEA-15 recruitment to the DISC (death-initiation signalling complex). Moreover, PEA-15 must be phosphorylated at Ser-116 to inhibit apoptosis. In the present study, we report that phosphorylation at Ser-104 blocks ERK binding to PEA-15 in vitro and in vivo, whereas phosphorylation at Ser-116 promotes its binding to FADD. We further characterize phospho-epitope-binding antibodies to these sites. We report that phosphorylation does not influence the distribution of PEA-15 between the cytoplasm and nucleus of the cell since all phosphorylated states are found predominantly in the cytoplasm. We propose that phosphorylation of PEA-15 acts as the switch that controls whether PEA-15 influences proliferation or apoptosis.

Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3 untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1-and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (AREmRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.Many mRNAs encoding proteins transiently required for inflammatory responses, cell proliferation, and intracellular signaling are labile due to AU-rich elements (AREs) in their 3Ј untranslated regions (UTRs) (14,21,57). ARE association by ELAV-like (embryonic lethal, abnormal vision) proteins, such as HuR, blocks ARE-mediated mRNA decay (AMD) (31). By contrast, association of proteins such as AUF1, tristetraprolin (TTP), BRF1 (butyrate-responsive factor-1), K-homology splicing regulatory protein (KSRP), ring finger K-homology domain 1 (RKHD1), polymyositisscleroderma 75-kDa antigen (PM-Scl75), or microRNA miR16 or miR289 with an ARE promotes AMD (6,8,12,18,24,34,43). The phosphorylation state of TTP, BRF1, and AUF1 affects AMD efficiency (3,37,51,56), indicating that signal transduction networks regulate this pathway.AUF1 has four protein isoforms-p37, p40, p42, and p45-generated by alternative pre-mRNA splicing (50). Based upon extensive biochemical studies of AUF1, we proposed an integrated, three-step model for induction of AMD by AUF1 via assembly of a trans-acting complex that targets the mRNA for degradation (52). The first step is dynamic AUF1 dimer binding to an ARE and formation of an oligomeric AUF1 complex (7, 52). Stabilizing ARE-binding proteins (AUBPs) may compete with AUF1 for binding to the ARE during this step, thus preventing AUF1 oligomerization and subsequent factor recruitment (25). Binding of AUF1 to an ARE then permits the second step involving recruitment of additional trans-acting factors including eukaryotic translation initiation factor eIF4G, poly(A)-binding protein, dual-functional heat shock/AUBPs Hsp/Hsc70 (27), and additional unknown proteins, forming a multisubunit AUF1-and signal tra...

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-␣) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues-Ser 15 , Ser 78 , and Ser 82 -by a number of kinases, including the mitogenactivated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.

No abstract

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array of MT1-MMP substrates, and MT1-MMP selective inhibitors have allowed for a more complete mapping of MT1-MMP biological functions. MT1-MMP has extensive sheddase activities, is both a positive and negative regulator of angiogenesis, can act intracellularly and as a transcription factor, and modulates immune responses. We presently examine the multi-faceted role of MT1-MMP in cancer, with a consideration of how the diversity of MT1-MMP behaviors impacts the application of MT1-MMP inhibitors.