Inhibition Of Extracellular Signal-regulated Kinase Signaling Research Articles

Regulation of Bidirectional Melanosome Transport by Organelle Bound MAP Kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.

Bidirectional signals transduced by DAPK?ERK interaction promote the apoptotic effect of DAPK

Death-associated protein kinase (DAPK) is a death domain-containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify the extracellular signal-regulated kinase (ERK) as a DAPK-interacting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser 735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. In a physiological apoptosis system where ERK-DAPK interplay is reinforced, downregulation of either ERK or DAPK suppresses such apoptosis. These results indicate that bidirectional signalings between DAPK and ERK may contribute to the apoptosis-promoting function of the death domain of DAPK.

Platelet‐Released Supernatant Increases Matrix Metalloproteinase‐2 Production, Migration, Proliferation, and Tube Formation of Human Umbilical Vascular Endothelial Cells

Local application of platelets represents a promising tool to enhance bone regeneration. New bone formation strictly requires blood vessel formation, a sequential process involving matrix degradation, migration, proliferation, and tube formation of endothelial cells. Here we investigated the impact of secreted granula products from activated platelets on endothelial cells, and determined the involvement of extracellular signal-regulated kinase (ERK) signaling. The effects of platelet-released supernatant on endothelial cells were investigated using in vitro models. Matrix metalloproteinase-2 (MMP-2) release, migration, proliferation, and tube formation of human umbilical vascular endothelial cells (HUVEC) were determined in response to platelet-released supernatant by gelatine zymography, Boyden chamber assay, 3[H]thymidine incorporation, and basement membrane assay, respectively. All experiments were performed in the presence of the ERK signaling inhibitor PD98059. ERK phosphorylation was detected by Western blot analysis. Incubation with platelet-released supernatant increased the production of MMP-2, migration, proliferation, and tube formation of HUVEC. Platelet-released supernatant also stimulated ERK phosphorylation in HUVEC. Inhibition of ERK signaling decreased platelet-released supernatant-stimulated endothelial cell proliferation, but not MMP-2 activity, migration, and the formation of capillary tubes. Our data suggest that secreted granula products from platelets can enhance different stages of blood vessel formation, and that ERK signaling is required to mediate the mitogenic effects of the supernatant. These findings support the hypothesis of a potential link between platelet activation and blood vessel formation during bone regeneration.