MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.     

The thioltransferase (glutaredoxin) 1 gene of fission yeast is regulated by Atf1 and Pap1

We previously isolated a gene encoding thioltransferase (TTase1) from the fission yeast Schizosaccharomyces pombe. Using a TTase-lacZ fusion plasmid, carrying a 666 bp region upstream of the translation initiation point, we found that expression of TTase1 was enhanced by metal ions, diamide and NO-generating S-nitroso-N-acetylpenicillamine (SNAP). In the present work, we examined the regulation of TTase1 expression using a series of deletion mutants and identified a negatively acting sequence between bp -469 and -339. Atf1 is required for basal expression of TTase1, and Pap1 is required for its inducible expression by mercuric chloride, diamide and SNAP. The -469 approximately -339 bp region is also responsible for mediating the inducible expression.     

The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.     

Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p

Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3′-untranslated region (3′UTR) or truncated 3′UTR fragments suggest that the first half of ATG16L1 3′UTR in the 5′ end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3′UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.     

The wheat protein kinase gene,TaPK3/, of the PKABA1 subfamily is differentially regulated in greening wheat seedlings

We have identified a new wheat PKABA1/-like protein kinase gene, TaPK3/, that is expressed in greening wheat seedlings. TaPK3 has high sequence homology (97% similarity with some sequence diversity at the 3' end) to the wheat PKABA1 protein kinase mRNA, which is upregulated by cold-temperature treatment, dehydration and abscisic acid (ABA). Use of a TaPK3 gene-specific probe has revealed that TaPK3 is differentially expressed with respect to PKABA1. TaPK3 mRNA accumulates in greening shoot tissue of wheat, but is not affected by dehydration, cold-temperature treatment or ABA. Based on sequence and expression differences, we conclude that expression of the PKABA1/-like protein kinases is not limited to stress responses.     

Aldehyde oxidase 1 gene is regulated by Nrf2 pathway

Aldehyde oxidase is a member of the molybd-flavo enzyme family that catalyzes the hydroxylation of heterocycles and the oxidation of aldehydes into corresponding carboxylic acids. Aldehyde oxidase-1 (AOX1) is highly expressed in liver and is involved in the oxidation of a variety of aldehydes and nitrogenous heterocyclic compounds, including anti-cancer and immunosuppressive drugs. However, the physiological substrates of AOX1 have not been identified, and it was unknown how the expression of AOX1 is regulated. Here, we found that the AOX1 gene is regulated by the Nrf2 pathway. Two Nrf2 binding consensus elements (antioxidant responsive element, ARE) are located in the 5' upstream region of the rat AOX1 gene. Molecular analyses using reporter transfection analysis, EMSA, and ChIP analysis show that Nrf2 binds to and strongly activates the rat AOX1 gene.     

Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.