The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization

Death-associated protein kinase 1 (DAPK-1) is a Ca²⁺/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.     

Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signaling

Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNA-mediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK(+/-) mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK+/+ counterparts, and overexpression of DAPK in DAPK(+/-) MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

Regulation of the specific DNA binding function of p53.

The DNA binding activity of p53 is required for its tumor suppressor function; we show here that this activity is cryptic but can be activated by cellular factors acting on a C-terminal regulatory domain of p53. A gel mobility shift assay demonstrated that recombinant wild-type human p53 binds DNA sequence specifically only weakly, but a monoclonal antibody binding near the C terminus activated the cryptic DNA binding activity stoichiometrically. p53 DNA binding could be activated by a C-terminal deletion of p53, mild proteolysis of full-length p53, E. coli dnaK (which disrupts protein-protein complexes), or casein kinase II (and coincident phosphorylation of a C-terminal site on p53). Activation of p53 DNA binding may be critical in regulation of its ability to arrest cell growth and thus its tumor suppressor function.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.