MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

Abstract

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.

Elevation of miR-21, through targeting MKK3, may be involved in ischemia pretreatment protection from ischemia–reperfusion induced kidney injury

“…The p38MAPK signaling pathway is activated by the upstream mitogen-activated protein kinase kinase 3 (MKK3), phosphorylates a variety of targets, and is involved in cell apoptosis, inflammation and fibrosis [7]. The MKK3-p38MAPK pathway is involved in apoptosis and inflammation of renal cells [8].…”

siRNA transfection in the barnacle Amphibalanus amphitrite larvae

“…In this state, p38 MAPK molecules do not have any functions. Once the larvae are stimulated, p38 MAPK will be phosphorylated by MAP kinase kinase 3 (MKK3), which then promotes larval settlement (Zhang et al, 2013). Using double transfection, the levels of p38 MAPK were decreased in lipofectin treatments and in treatments with 0.4 μl ml −1 siRNA and RNAfectin.…”

“…The procedures for adult barnacle collection and larvae culture are described in Zhang et al (2013). Nauplii developed to the VI stage after 3 days and transformed into cyprids after 4 days.…”

“…The protocols for total protein extraction, protein concentration determination and western blot are described in Zhang et al (2013). Antibodies against pp38 MAPK (Cell Signaling Technology, USA) and p38 MAPK (raised by He et al, 2012) were used to determine the level of pp38 MAPK and p38 MAPK, respectively.…”

“…So far, all the reported functional studie of genes, signal molecules and pathways in barnacle larval settlement have solely relied on chemical inhibitors, activators or analogues (Clare et al, 1995;He et al, 2012;Zhang et al, 2012Zhang et al, , 2013. However, these chemicals, which are often claimed to be highly specific and active, might have unexpected or unnoticed effects and thus raise serious concerns about the reliability of previous results.…”