Functional Analysis of Mitogen-Activated Protein Kinase-3 (MAPK3) and Its Regulation of the Promoter Region in Zebrafish Ming-Jyun Chiou, 1 Yi-Da Wang, 2 Ching-Ming Kuo, 1 Jian-Chyi Chen, 3,* and Jyh-Yih Chen 1,* Mitogen-activated protein kinase (MAPK) plays a pivotal role in intracellular actions in response to a variety of extracellular stimuli. Real-time reverse-transcription polymerase chain reaction analysis of MAPK3 tissue distri- bution in zebrafish showed significant differences in the fin and liver compared with muscle. A 1.2-kilobase (kb) pair and a 2.3-kb fragment of the 5 0 -flanking region displayed minimal promoter activity in the zebrafish liver (ZFL) and HeLa cell lines after treatment with insulin-like growth factors (IGF-I and IGF-II). Targeted knockdown of the MAPK3 gene by two antisense morpholino oligonucleotides revealed that although the zebrafish MAPK3 MO 1–targeted sequence was located at 5 0 untranslated region and the zebrafish MAPK3 MO 2–targeted sequence was located in the mature peptide region, similar results were shown in zebrafish for disruption of notochord development, with the whole body exhibiting distortion. From a comparative point of view, this study of the MAPK3 gene in zebrafish might not correlate well with previously published studies on mice. These molecular results suggest that MAPK3 plays an important role in whole-body development and is required for general embryonic development. Finally, MAPK3 may play important roles in fish cell growth. Introduction C ellular signal transduction in response to exter- nal stimuli by reversible protein phosphorylation con- stitutes one of the major mechanisms of cell communication. The initial tyrosine phosphorylation events are followed by ligand–receptor binding activities in a cascade of serine= threonine phosphorylation mediated by a sequence of serine= threonine kinases consisting of Ras, Raf, mitogen-activated protein (MAP), and S6 kinase. Among these factors, MAP kinases ( MAPKs) are crucial and ubiquitous in regulating im- portant cellular functions in response to mitogenic stimuli (Seger and Krebs, 1995; Robinson and Cobb, 1997). MAPKs are further characterized into three types: the first is p42=44 MAPK or extracellular receptor-mediated kinases (ERKs), the second consists of the stress-activated protein kinases (SAPKs)=c-Jun NH2 terminal kinases ( JNKs), and the third is p38. MAPKs are grouped into subfamilies on the basis of se- quence similarities, mechanisms of upstream regulation, and sensitivities to activation by different MAP kinase-ERK kinase (MEKs). Many MAPKs have been identified, including ERK5 and four p38-like kinases. Many different splice variants of JNK=SAPK are found in the brain. These variations that suggest different extracellular cues may produce stimulus- or tissue-specific responses through utilization of one of the MAPK patterns (Gupta et al., 1996; Jiang et al., 1996). The ac- tive translocation of MAPKs to the nucleus allows them to play a role like a transcription factor. Deletion of a nuclear export sequence from Xenopus MEK caused accumulation in the nucleus, which suggested that MEKs are normally rapidly exported when they enter the nucleus (Pomerance et al., 1996). The molecular biology of MAPKs is a complex conse- quence of a combination of many signal transduction path- ways and activities of kinases. Several common principles found in yeast should be similar for all MAPK pathways: (1) MAPKs are proline-directed kinases; (2) MAPK pathway components are often subject to regulation by multiple in- puts; (3) scaffolding proteins mediate pathway organization by MAPK regulation; and (4) membrane recruitment, oligo- merization, and phosphorylation regulate MAPK3. MAPK- activated protein kinases ( MAPKAPKs) are a family of serine= threonine protein kinases; they are substrates of MAPKs that play central roles in intracellular signal transduction pathways (Zu et al., 1998). There are three MAPK families: the ERKs, the c-Jun N-terminal kinase=SAPKs, and the p38 MAPKs. MAPKs are involved in the control of gene expres- sion, cell proliferation, and apoptosis (Robinson and Cobb, 1997). ERKs are activated by extracellular stimuli, such as 1 Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Jiaushi, Taiwan. 2 Department of Aquatic Biosciences, National Chiayi University, Chiayi, Taiwan. 3 Department of Biotechnology, Southern Taiwan University, Yung-Kang, Taiwan. *These authors should be considered as corresponding authors on this paper. DNA AND CELL BIOLOGY Volume 26, Number 11, 2007 ª Mary Ann Liebert, Inc. Pp. 781–790 DOI: 10.1089=dna.2007.0613 781