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No sunlight can reach the hadal trench, but some fishes dwelling there still have apparent eye morphology. However, whether they are capable of sensing light remains unknown. In this study, the eyes of the dominant hadal endemic snailfish Pseudoliparis swirei from the Mariana Trench were analyzed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A total of 2,088 proteins were identified in the eye proteome, most of which had at least one hit against public databases and could be mapped to 316 metabolic pathways. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways directly contributing to visual phototransduction were significantly enriched from the top 10% dominant proteins, implying abundant metabolic activities in the eye and it is still a functional visual organ. One rhodopsin was identified in the eye proteome. Sequence analysis indicated that it might have an absorption maximum at ∼480 nm and be sensitive to dim blue light. In addition, proteins that might contribute to extreme environment adaptation, such as heat shock proteins and chaperonin-containing T-complex protein 1, were also highly expressed in the eye. Overall, these results provide insights into the molecular mechanism underlying the vision of hadal snailfish and provide a useful database for further research. This article is protected by copyright. All rights reserved.

Citreamicin epsilon is a group of antitumor compounds produced by Streptomyces species. Cytotoxicology study of two diastereomers, citreamicin epsilon A and B, showed different apoptotic effects on PtK2 cells with IC50 (half-maximal inhibitory concentration) values of 0.086 and 0.025 mu M, respectively. Thus, we performed an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomic analysis to reveal the mechanism of cytotoxicity of citreamicin epsilon A and B in PtK2 cells. A total of 1,079 proteins were identified and quantified, among which 103 and 94 proteins displayed significant changes in expression levels after the treatment of citreamicin epsilon A and B, respectively. These significantly differentially expressed proteins (DEPs) were further annotated by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction analysis, which revealed the involvement of eight molecular pathways. Among them, expression trends of proteins involved in the nuclear transcription factor kappa B (NF-kappa B) pathway displayed the opposite between the two diastereomer treatments, indicating different modes of action of these two compounds. Citreamicin epsilon A treatment induced rapid activation of the NF-kappa B pathway, which might promote cell survival and resulted in lower toxicity. Our comparative proteomic analysis provided molecular evidence on the toxicity of two diastereomers compounds to cells, which may shed new light on future mechanism study of these antitumor compounds.

Barnacles represent one of the model organisms used for antifouling research, however, knowledge regarding the molecular mechanisms underlying barnacle cyprid cementation is relatively scarce. Here, RNA-seq was used to obtain the transcriptomes of the cement glands where adhesive is generated and the remaining carcasses of Megabalanus volcano cyprids. Comparative transcriptomic analysis identified 9060 differentially expressed genes, with 4383 upregulated in the cement glands. Four cement proteins, named Mvcp113k, Mvcp130k, Mvcp52k and Mvlcp1-122k, were detected in the cement glands. The salivary secretion pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes, implying that the secretion of cyprid adhesive might be analogous to that of saliva. Lysyl oxidase had a higher expression level in the cement glands and was speculated to function in the curing of cyprid adhesive. Furthermore, the KEGG enrichment analysis of the 352 proteins identified in the cement gland proteome partially confirmed the comparative transcriptomic results. These results present insights into the molecular mechanisms underlying the synthesis, secretion and curing of barnacle cyprid adhesive and provide potential molecular targets for the development of environmentally friendly antifouling compounds.

Barnacles, as major fouling organisms, have attracted more attentions. It is no doubt that the study on cement proteins is required to illustrate the mechanism of barnacle cementing. A cement protein defined as Aa-cp100k was characterized from Amphibalanus amphitrite in this study. The amino acid sequence of Aa-cp100k was shown a high similarity to other three barnacles including Megabalanus rosa (Mr-cp100k), Tetraclita japonica formosana (Tj-cp100k) and Pollicipes pollicipes (Pp-cp100k). Moreover, the localization of Aa-cp100k in the vacuoles of cyprid β secretory cells and the adult cement gland cells by immunofluorescence microscopy, indicating that Aa-cp100k existed in both cyprid and adult barnacle. Aa-cp100k from basal plate could be dissolved in urea buffer without high concentration of dithiothreitol (DTT), different from that in Megabalanus rosa, implying diverse possible roles of cp100k in cementing.

Diffusing fluid at a deep-sea hydrothermal vent creates rapid, acute physico-chemical gradients that correlate strongly with the distribution of the vent fauna. Two alvinocaridid shrimps, Alvinocaris longirostris and Shinkaicaris leurokolos occupy distinct microhabitats around these vents and exhibit different thermal preferences. S. leurokolos inhabits the central area closer to the active chimney, while A. longirostris inhabits the peripheral area. In this study, we screened candidate genes that might be involved in niche separation and microhabitat adaptation through comparative transcriptomics. The results showed that among the top 20% of overexpressed genes, gene families related to protein synthesis and structural components were much more abundant in S. leurokolos compared to A. longirostris. Moreover, 15 out of 25 genes involved in cellular carbohydrate metabolism were related to trehalose biosynthesis, versus 1 out of 5 in A. longirostris. Trehalose, a non-reducing disaccharide, is a multifunctional molecule and has been proven to act as a protectant responsible for thermotolerance in Saccharomyces cerevisiae. Putative positively selected genes involved in chitin metabolism and the immune system (lectin, serine protease and antimicrobial peptide) were enriched in S. leurokolos. In particular, one collagen and two serine proteases were found to have experienced strong positive selection. In addition, sulfotransferase-related genes were both overexpressed and positively selected in S. leurokolos. Finally, genes related to structural proteins, immune proteins and protectants were overexpressed or positively selected. These characteristics could represent adaptations of S. leurokolos to its microhabitat, which need to be confirmed by more evidence, such as data from large samples and different development stages of these alvinocaridid shrimps.

Alicella gigantea (Alicelloidae) is a scavenger with the largest body size among amphipods. It is a participant in the foodweb of deepsea ecosystem and distributed with vast bathymetric and geographic ranges. In this study, the mitochondrial genome of A. gigantea was completely assembled and characterized. The complete sequence has a total length of 16,851 bp, comprising the usual eukaryotic components, with 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 2 noncoding control regions (CRs). The gene rearrangement and reverse nucleotide strand bias of its mitochondrial genome are similar to those observed in the deepsea amphipod Eurythenes maldoror (Eurytheneidae), but different from the characters of Halice sp. MT-2017 (Dexaminoidea), an inhabitant of a deeper environment. Phylogenetic analysis indicates that A. gigantea occupies the basal branch of deepsea species—E. maldoror and Hirondellea gigas. This phylogeny supports the hypothesis that the evolution of hadal amphipods has undergone a transition from the abyssal depth. Compared to 41 available shallow water equivalents, the four accessible mitochondrial genomes from the deep sea, including the one produced in this study, show significantly fewer charged amino acids in the 13 PCGs, which suggests an adaption to the deepsea environment.

Citreamicins, members of the polycyclic xanthone family, are promising antitumor agents that are produced by Streptomyces species. Two diastereomers, citreamicin ε A (1) and B (2), were isolated from a marine-derived Streptomyces species. The relative configurations of these two diastereomers were determined using NMR spectroscopy and successful crystallization of citreamicin ε A (1). Both diastereomers showed potent cytotoxic activity against HeLa (cervical cancer) and HepG2 (hepatic carcinoma) cells with IC50 values ranging from 30 to 100 nM. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed that citreamicin ε A (1) induced cellular apoptosis, and Western blot analysis showed that apoptosis occurred via activation of caspase-3. The 2,7-dichlorofluorescein diacetate assay indicated that citreamicin ε substantially increased the intracellular concentration of reactive oxygen species (ROS). To confirm the hypothesis that citreamicin ε induced apoptosis through an increase in the intracellular ROS concentration, the oxidized products, oxicitreamicin ε A (3) and B (4), were obtained from a one-step reaction catalyzed by Ag2O. These products, with a reduced capacity to increase the intracellular ROS concentration, exhibited a significantly weakened cytotoxicity in both HeLa and HepG2 cells compared with that of citreamicin ε A (1) and B (2).

N-acetylneuraminic acid (Neu5Ac) based novel pharmaceutical agents and diagnostic reagents are highly required in medical fields. However, N-acetylneuraminate lyase（NAL）for Neu5Ac synthesis is not applicable for industry due to its low catalytic efficiency. In this study, we biochemically characterized a deep-sea NAL enzyme (abbreviated form: MyNal) from a symbiotic Mycoplasma inhabiting the stomach of a deep-sea isopod, Bathynomus jamesi. Enzyme kinetic studies of MyNal showed that it exhibited a very low Km for both cleavage and synthesis activities compared to previously described NALs. Though it favors the cleavage process, MyNal out-competes the known NALs with respect to the efficiency of Neu5Ac synthesis and exhibits the highest kcat/Km values. High expression levels of recombinant MyNal could be achieved (9.56 mol L−1 culture) with a stable activity in a wide pH (5.0–9.0) and temperature (40–60 °C) range. All these features indicated that the deep-sea NAL has potential in the industrial production of Neu5Ac. Furthermore, we found that the amino acid 189 of MyNal (equivalent to Phe190 in Escherichia coli NAL), located in the sugar-binding domain, GX189DE, was also involved in conferring its enzymatic features. Therefore, the results of this study improved our understanding of the NALs from different environments and provided a model for protein engineering of NAL for biosynthesis of Neu5Ac.

The bryozoan Bugula neritina has a biphasic life cycle that consists of a planktonic larval stage and a sessile juvenile/adult stage. The transition between these two stages is crucial for the development and recruitment of B. neritina. Metamorphosis in B. neritina is mediated by both the nervous system and the release of developmental signals. However, no research has been conducted to investigate the expression of neuropeptides (NP)/peptide hormones in B. neritina larvae. Here, we report a comprehensive study of the NP/peptide hormones in the marine bryozoan B. neritina based on in silico identification methods. We recovered 22 transcripts encompassing 11 NP/peptide hormone precursor transcript sequences. The transcript sequences of the 11 isolated NP precursors were validated by cDNA cloning using gene-specific primers. We also examined the expression of three peptide hormone precursor transcripts (BnFDSIG, BnILP1, BnGPB) in the coronate larvae of B. neritina, demonstrating their distinct expression patterns in the larvae. Overall, our findings serve as an important foundation for subsequent investigations of the peptidergic control of bryozoan larval behavior and settlement.

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.

Bathymodiolid mussels dominate hydrothermal vents, cold methane/sulfide-hydrocarbon seeps and other sites of organic enrichment. Here, we aimed to explore the innate immune system and detoxification mechanism of the deep sea mussel Bathymodiolus platifrons collected from a methane seep in the South China Sea. We sequenced the transcriptome of the mussels’ gill, foot and mantle tissues and generated a transcriptomic database containing 96,683 transcript sequences. Based on GO and KEGG annotations, we reported transcripts that were related to the innate immune system, heavy metal detoxification and sulfide metabolic genes. Our in-depth analysis on the isoforms of peptidoglycan recognition protein (PGRP) that have different cellular location and potentially differential selectivity towards peptidoglycan (PGN) from gram-positive and gram-negative bacteria were differentially expressed in different tissues. We also reported a potentially novel form of metallothionein and the production of phytochelatin in B. platifrons, which has not been reported in any of its coastal relative Mytilus mussel species. Overall, the present study provided new insights into heavy metal and sulfide metabolism in B. platifrons and can be served as the basis for future molecular studies on host-symbiont interactions in cold seep mussels.

The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands.

As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus) amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein), we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study.

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label-free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron-mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.

Marine biofouling refers to the unwanted accumulation of fouling organisms, such as barnacles, on artificial surfaces, resulting in severe consequences for marine industries. Meleagrin is a potential nontoxic antifoulant that is isolated from the fungus Penicillium sp.; however, its mechanistic effect mode of action on larval settlement remains unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the effect of meleagrin on the proteomic expression profile of cyprid development and aging in the barnacle Balanus amphitrite . Fifty proteins were differentially expressed in response to treatment with meleagrin, among which 26 proteins were associated with cyprid development/aging and 24 were specifically associated with the meleagrin treatment. The 66 proteins that were associated with aging only remained unaltered during exposure to meleagrin. Using KEGG analysis, those proteins were assigned to several groups, including metabolic pathways, ECM-receptor interactions, and the regulation of the actin cytoskeleton. Among the 24 proteins that were not related to the development/aging process, expression of the cyprid major protein (CMP), a vitellogenin-like protein, increased after the meleagrin treatment, which suggested that meleagrin might affect the endocrine system and prevent the larval molting cycle. With the exception of the chitin binding protein that mediates the molting process and ATPase-mediated energy processes, the majority of proteins with significant effects in previous studies in response to cyprid treatment with butenolide and polyether B remained unchanged in the present study, suggesting that meleagrin may exhibit a different mechanism.

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing.

The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts.

Butenolide is a very promising antifouling compound that inhibits ship hull fouling by a variety of marine organisms, but its antifouling mechanism was previously unknown. Here we report the first study of butenolide's molecular targets in three representative fouling organisms. In the barnacle Balanus (=Amphibalanus) amphitrite, butenolide bound to acetyl-CoA acetyltransferase 1 (ACAT1), which is involved in ketone body metabolism. Both the substrate and the product of ACAT1 increased larval settlement under butenolide treatment, suggesting its functional involvement. In the bryozoan Bugula neritina, butenolide bound to very long chain acyl-CoA dehydrogenase (ACADVL), actin, and glutathione S-transferases (GSTs). ACADVL is the first enzyme in the very long chain fatty acid β-oxidation pathway. The inhibition of this primary pathway for energy production in larvae by butenolide was supported by the finding that alternative energy sources (acetoacetate and pyruvate) increased larval attachment under butenolide treatment. In marine bacterium Vibrio sp. UST020129-010, butenolide bound to succinyl-CoA synthetase β subunit (SCSβ) and inhibited bacterial growth. ACAT1, ACADVL, and SCSβ are all involved in primary metabolism for energy production. These findings suggest that butenolide inhibits fouling by influencing the primary metabolism of target organisms.

The involvement of cyclin-dependent kinase-5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron-fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent-resistant fraction made up of nuclear matrix, membrane-bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10-mediated apoptosis occurred via a caspases-independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent-insoluble fraction may be a crucial pathological event to triggering neuronal cell death.

In the nervous system, Cdk5 and its neuronal activator p35 are involved in the control of various activities, including neuronal differentiation and migration. Recently, we have reported that p35 is a microtubule-associated protein that regulates microtubule dynamics ( Hou, Z., Li, Q., He, L., Lim, H. Y., Fu, X., Cheung, N. S., Qi, D. X., and Qi, R. Z. (2007) J. Biol. Chem. 282, 18666-18670 ). Here we present two regulatory modes of p35 function as a microtubule-associated protein. First, p35 is Ca(2+)-dependent calmodulin (CaM)-binding protein. The CaM- and microtubule binding domains are localized to overlapping regions at the N terminus of p35. Within the CaM-binding region, Ala substitution for Trp-52 abolishes the CaM-binding activity, corroborating specific CaM-binding of p35. Furthermore, CaM blocks p35 association with microtubules in a Ca(2+)-specific manner, suggesting that p35 may be involved in the Ca(2+)/CaM-mediated inhibition of microtubule assembly. Second, p35 phosphorylation by Cdk5 interferes with the microtubule-binding and polymerizing activities of p35. Using a mutational approach, we found that only phosphorylation at Thr-138, one of the two residues primarily phosphorylated in vivo, inhibits the polymerizing activity. In PC12 cells, expression of p35 promotes nerve growth factor-induced neurite outgrowth under a Cdk5 inhibitory condition. Such p35 activity is impaired by the phosphomimetic mutation of Thr-138. These data suggest that Thr-138 phosphorylation plays a critical role in the control of the p35 functions in microtubule assembly and neurite outgrowth.

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.

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.