Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk. This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro. Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase. We employed the yeast twohybrid system to identify interaction partners of Dlk that might serve as regulators or targets. Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis. Complex formation between Dlk and Par-4 was con®rmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo. The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 ± 417, rather than to the leucine zipper. Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin ®laments. These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis.
One of the pathological lesions in Alzheimer's disease (AD) is the amyloid or senile plaque. The plaque core is predominantly made up of amyloid beta peptide (A beta), a 42-43 amino acid peptide derived from amyloid precursor protein (APP). APP is a membrane bound glycoprotein which is expressed ubiquitously in many cells. Although normal or pathological functions for APP are not well understood, several observations suggest that APP may play a role in cellular stress and inflammation at the endothelial cell/vascular barrier. APP is found in platelets and endothelial cells, it can inhibit a blood coagulation factor, and secreted APP can be neuroprotective. Changes in expression of APP during cellular stress or inflammation may contribute to pathological deposition of A beta. In the present studies, expression of APP in human endothelial cells was examined following heat shock. In human umbilical vein endothelial cells (HUVECs) exposed to 42 degrees C for 30 min, there was a five- to eight-fold increase in APP mRNA levels which peaked at 4 hr. The increase in APP mRNA was followed by an increase in APP protein immunoreactivity in the cytoplasm in a perinuclear Golgi-like region, and in discrete granular cytoplasmic structures. Immunoblot analysis of APP in the cell media found a transient increase in APP which peaked at 1 hr after heat shock. These results suggest that cellular stress induces the secretion of APP from endothelial cells followed by a subsequent increase in APP mRNA and protein synthesis. The upregulation of APP mRNA and protein supports a cellular stress role for APP.
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