Mitogen-activated protein kinase (MAPK) is activated in cytoplasm in response to extracellular signals and then is translocated to nucleus. A directed activator for MAPK, MAPK kinase (MAPKK), stays in cytoplasm to transmit the signal from the plasma membrane to MAPK. Here we show that MAPKK contains a short amino acid sequence in the N-terminal region (residues 32-44), which acts as a nuclear export signal (NES) and thus is required for cytoplasmic localization of MAPKK. This NES sequence of MAPKK, like that of protein kinase inhibitor of cAMP-dependent protein kinase or Rev, is rich in leucine residues, which are crucial for the NES activity. Furthermore, the NES peptide of protein kinase inhibitor, as well as the NES peptide of MAPKK, inhibited the nuclear export of ovalbumin conjugated to the NES peptide of MAPKK. These results may suggest a common mechanism of nuclear export using a general leucine-rich NES.Mitogen-activated protein kinase (MAPK) 1 is activated in response to a wide variety of extracellular stimuli (1-4) and functions as one of several important mediators of signal transductions that control cell proliferation (5-10), cell differentiation (7,11,12), and early embryonic development (13-15). Activation of MAPK requires its dual phosphorylation on threonine and tyrosine residues catalyzed by MAPK kinase (MAPKK), a dual-specificity protein kinase (16,17). MAPKK exists in cytoplasm (18 -20) and is activated by serine phosphorylation (21-23) catalyzed by an upstream serine/threonine kinase, such as Raf-1 (24 -26), which may be activated near the plasma membrane (27-30). Thus, MAPKK is a key intermediate in the MAPK cascade, and cytoplasmic localization of MAPKK may be important for the proper signal transduction of the MAPK cascade. In fact, MAPK is first activated in cytoplasm through activation of MAPKK and then translocated to the nucleus (18,31,32). However, it remains unclear how cytoplasmic localization of MAPKK is achieved.The first identification of nuclear export signal (NES) (33-38) was recently done in studies characterizing two specific proteins (human immunodeficiency virus, type I-coded Rev protein and inhibitor (PKI) of cAMP-dependent protein kinase) that rapidly shuttle between the nucleus and the cytoplasm. The NES sequences in Rev and PKI are both rich in hydrophobic residues, in which three leucine residues are critical for nuclear export activity (33)(34)(35)(36)(37)(38).During the course of experiments originally designed to elucidate the mechanism of the nuclear translocation of MAPK, we found that MAPKK has in its N-terminal region a short sequence that regulates its subcellular distribution and that the sequence has an NES activity. This NES sequence of MAPKK, like the NES of PKI or Rev, is rich in leucine residues, which were found to be crucial for its NES activity. Furthermore, the NES of PKI and the NES of MAPKK competed with each other. These results may suggest a common mechanism of nuclear export using a general leucine-rich NES. MATERIALS AND METHODSDNA Construct...

Mitogen-activated protein kinase (MAPK) kinase (MAPKK, also known as MEK), a direct activator for MAPK/extracellular signal-regulated kinase, localizes to the cytoplasm excluded from the nucleus during signal transmission. This nuclear exclusion of MAPKK is directed by its nuclear export signal (NES), but its physiological significance has been unknown. We have found that disruption of the NES dramatically potentiates the ability of constitutively active MAPKK to induce morphological changes and malignant transformation of fibroblastic cells. Readdition of the NES sequence reversed the effects induced by the NES disruption. Moreover, we observed that a dramatic increase of activated MAPK in the nucleus was induced by the NESdisrupted MAPKK and that coexpression of MAPK phosphatase-1 (CL-100) or a kinase negative form of MAPK counteracted the phenotypes induced by the NES-disrupted MAPKK, indicating the crucial role of MAPK in the responses. These findings reveal a novel regulatory role of the NES of MAPKK that may be essential for proper signal transductions.Mitogen-activated protein kinase (MAPK), 1 also known as extracellular signal-regulated kinase, is commonly activated by a wide variety of extracellular stimuli (reviewed in Refs. 1-5). Activation of MAPK requires its dual phosphorylation on threonine and tyrosine residues catalyzed by a direct activator, MAPK kinase (MAPKK, also known as MEK) (reviewed in Refs. 3, 4, and 6). MAPKK is activated by phosphorylation on serine residues by MAPKK kinase such as the proto-oncogenes Raf-1 and Mos (7-12). These three kinases form a linear pathway called the MAPK cascade, which is one of the central intracellular signaling pathways that control cell proliferation, cell differentiation, and early embryonic development.Events leading to the activation of the MAPK cascade are initiated at plasma membrane with ligand binding to various receptors. Binding of ligand to the receptor tyrosine kinase results in the activation of the proto-oncogene product Ras (reviewed in Ref. 13). The activated Ras recruits Raf-1 to the plasma membrane where the activation of Raf-1 occurs (14 -20). Then the activated Raf-1, as MAPKK kinase, phosphorylates and activates MAPKK, which in turn activates MAPK in the cytoplasm. These three kinases undergo multiple phosphorylation reactions in the cytoplasm, whereas the terminal component of the three kinases, MAPK, is translocated from the cytoplasm to the nucleus (21-23) where MAPK phosphorylates several nuclear targets such as Elk-1, Myc, and Myb (reviewed in Refs. 24 and 25). Thus, the MAPK cascade constitutes a functional unit that links cytoplasmic signaling events to nuclear events.MAPKK stays in the cytoplasm during signal transmission (23,26,27). We have previously reported that MAPKK has in its near N-terminal region a short amino acid sequence that acts as a nuclear export signal (NES) and that this NES directs cytoplamic localization or nuclear exclusion of MAPKK (28). The concept of NES initially emerged from studies characterizing two ...

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MTCBP-1 was identified as a protein that binds