Anaplastic Lymphoma Kinase Is Required for Neurogenesis in the Developing Central Nervous System of Zebrafish

10.1371/journal.pone.0063757PLoS ONE85

Structural Analysis of the MAP Kinase ERK2 and Studies of MAP Kinase Regulatory Pathways

This chapter primarily highlights structure–function studies performed collaboratively between the Goldsmith and Cobb laboratories at the University of Texas Southwestern Medical Center in Dallas and includes experiments examining the relationship of MEKKl to the MAP kinase pathway and potential feedback mechanisms in the pathway. Mechanisms regulating the MAP kinase pathway are complicated and inactidvation methods plentiful. Receptor tyrosine kinases regulate the pathway through Ras; heterotrimeric G protein-coupled receptors also use Ras to activate the pathway, although there may also be Ras-independent mechanisms. The MAP kinase pathway is stimulated by numerous hormones, growth factors, and oncogene products including Ras and contributes to their spectrum of actions. The MAP kinases, however, are pleiotropic, phosphorylating many substrates throughout the cell. This pathway has been repeated several times in yeast and mammalian cells, although mechanisms regulating the similar but parallel cascades are sketchier. There are three low-activity forms of each enzyme, the unphosphorylated protein and the two singly phosphorylated forms, that contain phosphate on only tyrosine or only threonine. These two singly phosphorylated ERKs have little more protein kinase activity than unphosphorylated proteins. Because ERKl and ERK2 can autophosphorylate on tyrosine, the form containing only threonine phosphate may, in as yet undefined circumstances, be able to reactivate itself through autophosphorylation. The three-dimensional structure of ERK2 contains the two-domain organization found in all protein kinases whose structures have been determined thus far. The smaller N-terminal domain provides many contacts for ATP, and the larger C-terminal domain contains the major determinants for protein substrate interactions. It is very important to identify the unique features, if any, of these enzyme pairs. It is interesting that phosphorylation by MAP kinase had so little effect on MEK activity, because phosphorylation of MEKl by cdc2 at a site (S286) close to the MAP kinase site (T292) in the C-terminal insert greatly inhibits MEKl activity

Map Kinases Erk1 And Erk2: Pleiotropic Enzymes In A Ubiquitous Signaling Network

MAP kinase is used to refer to at least two distinct proteins, known as the extracellular signal-regulated protein kinases ERK 1 and ERK2. The discussion of proteins that are regulated by ERKs points to the pleiotropic nature of signaling pathways regulated by this family of protein kinases. Relatively, few data are yet in hand to prove that any of these proteins are substrates for these enzymes in vivo. The impact of these enzymes on cell function can be deduced from the recent experiments using mutated enzymes. The ERKl phosphorylation site and lysine mutants have proved useful dominant inhibitors. In Jurkat cells, the mutants inhibit induction of the cytokine IL-2. It was found that ERKl and ERK2 mutants block the ability of ras, serum, and phorbol ester to induce transcription from a TPA response element. ERK2 mutants prevent proliferation caused by activated Raf EGF or small tau antigen. The ubiquitous MAP kinases are activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells. Their activation has been linked to the transition from G0 to G1 in the cell cycle and to the induction of differentiated phenotypes. These enzymes are essential components of a universal protein kinase cascade implicated in the control of many cellular processes

Overexpression of <i>alk</i> promotes cell proliferation and affects neurogenesis.

<p><b>(A,B)</b> Confocal sections of HSE:<i>cfp</i> embryos. Sib (A) and Tg+ (B) embryos show no difference in number and distribution of pH 3 and HuC/D positive cells. <b>(C)</b> Y-axis indicates numbers of pH 3 positive cells counted in a 50 µm confocal stack of the hindbrain. Mean ± SEM, n = 10 embryos in each group. Sib and Tg+ HSE:<i>cfp</i> embryos were not significantly different (p = 0.84). Sib and Tg+ embryos of both <i>alk</i>:HSE:<i>cfp</i> lines were significantly different (***p<0.001). Unpaired two tailed t-test. <b>(D–G)</b> Confocal sections of <i>alk</i>:HSE:<i>cfp</i>¹ embryos. Sib (D) and Tg+ (E–G, from three different embryos) had different neural tube shapes. Dividing cells (pH 3, green) and neurons (HuC/D, red) in Tg+ embryos (E–G) were mispositioned (arrowheads), with aberrant patterns. <b>(E′)</b> High magnification of the boxed area in (E). Asterisk labels 4^th ventricle. Arrows label small cavities found in the neural tube. <b>(H,I)</b> Confocal sections of <i>alk</i>:HSE:<i>cfp</i>¹ embryos, with BrdU labelled cells in S-phase. In Sib (H), BrdU positive cells occupy a region between dividing cells and neurons that exited the cell cycle, in a pattern complementary to pH 3 and HuC/D in (A,B,D). In Tg+ (I), BrdU positive cells were randomly positioned. Smaller dimension of samples in (H,I) might be due to HCl treatment in the experiment procedure. <b>(J,K)</b> Manual sections of embryos after <i>in situ</i> hybridization showed expanded <i>ccnd1</i> expressions in Tg+ (K) in comparison to Sib embryos (J). Sib, transgenic negative siblings. Tg+, transgenic positive embryos. All images are sections perpendicular to neural tube. Scale bars: 50 µm.</p

Expression of zebrafish <i>alk</i>.

<p><b>(A)</b> RT-PCR of cDNA at different embryonic stages. <i>gapdh</i> is used as loading control. <b>(B)</b> RT-PCR of cDNA from different adult tissues. <i>beta-actin</i> is used as loading control. <b>(C)</b> Lateral view of whole embryos at 24 hpf with dorsal up and anterior to the left. Insert shows dorsal view of head region. <b>(D–G)</b> Transverse sections of embryos at 24 hpf, at forebrain (D), midbrain (E), rhombomere 2 (r2; F) and r5 (G) levels, corresponding to positions indicated by lines in (C). <b>(H)</b> Lateral view of whole embryo at 48 hpf with dorsal up and anterior to the left. Insert shows dorsal view of head region. <b>(I–K)</b> Transverse sections of embryos at 48 hpf at forebrain (I), midbrain (J), and r5 (K), corresponding to positions indicated by lines in (D). Scale bars: C,H, 200 µm; D–E,I–K, 100 µm.</p

Knock-down of <i>alk</i> impairs neuronal differentiation.

<p><b>(A–L)</b><i>In situ</i> hybridization of neuronal marker genes with wild-type (A,C,E,G,I,K), and <i>alk</i> Spl-MO injected embryos (B,D,F,H,J,L) at 22 hpf. Images in first row show dorsal views of head region with anterior to the left. <b>(A′–L′,A″ –L″)</b> Transverse cross sections at the level of r2 (A′–L′) and r5 (A″ –L″) in WT embryos or <i>alk</i> Spl-MO injected morphants. <b>(A,A′A″,B,B′B″)</b><i>her6</i> expression was unchanged in <i>alk</i> morphants compared to WT. <b>(C,C′,C″,D,D′,D″)</b><i>neurog1</i> expression was unchanged or only slightly reduced in morphants. <b>(E,E′,E″,F,F′,F″)</b><i>neurod4</i> expression was strongly reduced in morphants in several regions including the hindbrain. <b>(G,G′,G″,H,H′,H″)</b> Similarly, <i>ascl1b</i> expression in <i>alk</i> morphants was also significantly reduced. <b>(I,I′,I″,J,J′,J″)</b><i>dla</i> expression in <i>alk</i> morphants was also reduced. <b>(K,K′,K″,L,L′,L″)</b> Glia marker <i>gfap</i> expression was unchanged. Scale bars: 50 µm. Lines at r2 and r5 indicate levels of cross sections.</p

Knock-down of <i>alk</i> does not affect proliferation but induces apoptosis in the hindbrain.

<p><b>(A–C)</b> Confocal sections of 22 hpf embryos after immunostaining with pH 3 (green) and TUNEL (red). Compared to WT (A), both <i>alk</i> ATG-MO injected (B) or <i>alk</i> Spl-MO injected (C) embryos show pH 3 positive cells at normal positions, but more TUNEL positive cells are evident. Note that the size of neural tube in (B,C) is smaller than in (A). <b>(D)</b> Y-axis indicates numbers of pH 3 positive cells in a 50 µm thick confocal stack of hindbrain. Numbers are not significantly different in <i>alk</i> ATG-MO or <i>alk</i> Spl-MO from WT. <b>(E)</b> Y-axis indicates numbers of TUNEL positive cells in the same samples. Both <i>alk</i> ATG-MO and <i>alk</i> Spl-MO numbers were different from that in WT with high significance (***p<0.001). Mean ± SEM, n = 5 embryos in each group. Unpaired two tailed t-test. Scale bars: 50 µm.</p