viral-mediated-labeling-transplantation-medial-ganglionic-eminence

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for <em>In Vivo</em> Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

University of California San Francisco

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and possesses apoptotic and tumor-suppressive functions. However, it is unclear whether DAPK elicits apoptosis-independent activity to suppress tumor progression. We show that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These effects are mainly mediated by an inhibitory role of DAPK in talin head domain association with integrin, thereby suppressing the integrin-Cdc42 polarity pathway. We present evidence indicating that the antimigratory effect of DAPK represents a mechanism through which DAPK suppresses tumors. First, DAPK can block migration and invasion in certain tumor cells that are resistant to DAPK-induced apoptosis. Second, using an adenocarcinoma cell line and its highly invasive derivative, we demonstrate DAPK level as a determining factor in tumor invasiveness. Collectively, our study identifies a novel function of DAPK in regulating cell polarity during migration, which may act together with its apoptotic function to suppress tumor progression.

The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway.

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.

The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR.

Interleukin-1β (IL-1β) is critical for inflammation and control of infection. The production of IL-1β depends on expression of pro-IL-1β and inflammasome component induced by inflammatory stimuli, followed by assembly of inflammasome to generate caspase-1 for cleavage of pro-IL-1β. Here we show that tumor suppressor death-associated protein kinase (DAPK) deficiency impaired IL-1β production in macrophages. Generation of tumor necrosis factor-α in macrophages, in contrast, was not affected by DAPK knockout. Two tiers of defects in IL-1β generation were found in DAPK-deficient macrophages: decreased pro-IL-1β induction by some stimuli and reduced caspase-1 activation by all inflammatory stimuli examined. With a normal NLRP3 induction in DAPK-deficient macrophages, the diminished caspase-1 generation is attributed to impaired inflammasome assembly. There is a direct binding of DAPK to NLRP3, suggesting an involvement of DAPK in inflammasome formation. We further illustrated that the formation of NLRP3 inflammasome in situ induced by inflammatory signals was impaired by DAPK deficiency. Taken together, our results identify DAPK as a molecule required for full production of IL-1β and functional assembly of the NLRP3 inflammasome. In addition, DAPK knockout reduced uric acid crystal-triggered peritonitis, suggesting that DAPK may serve as a target in the treatment of IL-1β-associated autoinflammatory diseases.

Tumor suppressor death-associated protein kinase is required for full IL-1β production.

Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear.

Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594-604, Δ605-612 and Δ626-634) and the C-terminal tail (Δ14; deletion of 1164-1177). The expression of Δ594-604 and Δ605-612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626-634 and Δ1164-1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594-604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594-604) regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177.

Article	Year
The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway. The Journal of cell biology\| PubMed ID: 16476779	2006
The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR. Molecular cell\| PubMed ID: 17803936	2007
Tumor suppressor death-associated protein kinase is required for full IL-1β production. Blood\| PubMed ID: 21041719	2011
Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. PloS one\| PubMed ID: 22768143	2012
The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177. Bioscience reports\| PubMed ID: 24993645	2014

Pei-Rung Wu

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