5 research outputs found
ZIPK: A Unique Case of Murine-Specific Divergence of a Conserved Vertebrate Gene
Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique proteinâprotein interaction that enables conservation of cellular function
New Modularity of DAP-Kinases: Alternative Splicing of the DRP-1 Gene Produces a ZIPk-Like Isoform
DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase
(DAP-kinase) family, which function in different settings of cell death
including autophagy. DAP kinases are very similar in their catalytic domains but
differ substantially in their extra-catalytic domains. This difference is
crucial for the significantly different modes of regulation and function among
DAP kinases. Here we report the identification of a novel alternatively spliced
kinase isoform of the DRP-1 gene, termed DRP-1β. The
alternative splicing event replaces the whole extra catalytic domain of DRP-1
with a single coding exon that is closely related to the sequence of the extra
catalytic domain of ZIPk. As a consequence, DRP-1β lacks the calmodulin
regulatory domain of DRP-1, and instead contains a leucine zipper-like motif
similar to the protein binding region of ZIPk. Several functional assays proved
that this new isoform retained the biochemical and cellular properties that are
common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and
activation of membrane blebbing and autophagy. In addition, DRP-1β also
acquired binding to the ATF4 transcription factor, a feature characteristic of
ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like
isoform. DRP-1β is highly conserved in evolution, present in all known
vertebrate DRP-1 loci. We detected the corresponding mRNA and
protein in embryonic mouse brains and in human embryonic stem cells thus
confirming the in vivo utilization of this isoform. The
discovery of module conservation within the DAPk family members illustrates a
parsimonious way to increase the functional complexity within protein families.
It also provides crucial data for modeling the expansion and evolution of DAP
kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely
evolved from their ancient ancestor gene DAPk by two gene duplication events
that occurred close to the emergence of vertebrates
Sequence variation in PPP1R13L results in a novel form of cardioâcutaneous syndrome
Dilated cardiomyopathy (DCM) is a lifeâthreatening disorder whose genetic basis is heterogeneous and mostly unknown. Five Arab Christian infants, aged 4â30 months from four families, were diagnosed with DCM associated with mild skin, teeth, and hair abnormalities. All passed away before age 3. A homozygous sequence variation creating a premature stop codon at PPP1R13L encoding the iASPP protein was identified in three infants and in the mother of the other two. Patientsâ fibroblasts and PPP1R13Lâknocked down human fibroblasts presented higher expression levels of proâinflammatory cytokine genes in response to lipopolysaccharide, as well as Ppp1r13lâknocked down murine cardiomyocytes and hearts of Ppp1r13lâdeficient mice. The hypersensitivity to lipopolysaccharide was NFâÎşBâdependent, and its inducible binding activity to promoters of proâinflammatory cytokine genes was elevated in patientsâ fibroblasts. RNA sequencing of Ppp1r13lâknocked down murine cardiomyocytes and of hearts derived from different stages of DCM development in Ppp1r13lâdeficient mice revealed the crucial role of iASPP in dampening cardiac inflammatory response. Our results determined PPP1R13L as the gene underlying a novel autosomalârecessive cardioâcutaneous syndrome in humans and strongly suggest that the fatal DCM during infancy is a consequence of failure to regulate transcriptional pathways necessary for tuning cardiac threshold response to common inflammatory stressors