An essential role of Pak1 phosphorylation of SHARP in Notch signaling (original) (raw)
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Oncogene, 2002
The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have cloned a novel human PAK family kinase that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. We have shown that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly exp...
p21-activated kinases: three more join the Pak
The international journal of biochemistry & cell …, 2002
The p21-activated kinases (Paks) are serine/threonine protein kinases that bind to and, in some cases, are stimulated by activated forms of the small GTPases, Cdc42 and Rac. With the recent discovery of several novel isoforms, Paks are now categorized into two subgroups based on architectural similarities. The Group I Paks (Pak1, Pak2, Pak3) have been studied in greater detail and shown to be involved in the regulation of cellular processes such as gene transcription, cell morphology, motility, and apoptosis. Here we summarize recent findings that shed light on the newly recognized Group II Paks (Pak4, Pak5, Pak6) and review both similarities and differences between kinases of the two Pak subgroups.
Molecular Cloning of a New Member of the p21-Cdc42/Rac-activated Kinase (PAK) Family
Journal of Biological Chemistry, 1995
A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65 PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). The purified serine/threonine kinase p65 PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform -PAK, which shares 79% amino acid identity with the previously described ␣-isoform. Their mRNAs are differentially expressed in the brain, with ␣-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the ␣and -PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific -PAK peptide sequence was obtained from rat brainpurified p65 PAK. Recombinant ␣and -PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of ␣-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant -PAK and brain p65 PAK , which is observed upon kinase activation does not occur with recombinant ␣-PAK.
A Novel Regulator of p21-activated Kinases
Journal of Biological Chemistry, 1998
Proteins of the p21-activated kinase (Pak) family have been implicated in the regulation of gene expression, cytoskeletal architecture, and apoptosis. Although the ability of Cdc42 and Rac GTPases to activate Pak is well established, relatively little else is known about Pak regulation or the identity of Pak cellular targets. Here we report the identification of two closely related Pak3binding proteins, possibly arising from alternative splicing, designated p50 and p85 Cool-1 (cloned out of library). Both isoforms of Cool contain a Src homology 3 domain that directly mediates interaction with Pak3 and tandem Dbl homology and pleckstrin homology domains. Despite the presence of the Dbl homology-pleckstrin homology motif, a characteristic of Rho family activators, activation of Cdc42 or Rac by Cool is not detectable. Instead binding of p50 Cool-1 , but not p85 Cool-1 , to Pak3 represses its activation by upstream activators such as the Dbl oncoprotein, indicating a novel mechanism of regulation of Pak signaling.
2012
Ammonium persulfate bp Base pair BSA Bovine serum albumin CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate Ci Curie CIP Calf Intestinal Alkaline Phosphatase D-MEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid FCS fetal calf serum FP Fluorescent Protein g Gram h hour HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid H 2 O 2 Hydrogen peroxide IgG Immunoglobulin G kb Kilobase pairs kDa Kilodalton l Liter LacZ β-galactosidase LB Lysogeny broth M Mol mA Milliampere mg Milligram min Minute ml Millilitre mM Millimolar mRNA Messenger RNA nm Nanometer NP-40 nonyl phenoxypolyethoxylethanol OD Optic density p pico PBS Phosphate buffered saline PMSF phenylmethylsulfonyl fluoride PVDF Polyvinylidene Fluoride RNase Ribonuclease rpm Rounds per minute RT Room temperature SDS Sodium dodecyl sulfate SOB Super Optimal Broth medium SCC Saline-sodium citrate TAE Tris-acetate buffer TE Tris-EDTA buffer TEMED Tetramethylethylenediamine TWEEN Polyoxyethylene (20) sorbitan monolaurate U Units UV Ultraviolet Light µg Microgram µl Microliter 1. INTRODUCTION 1 The embryonic development of higher organisms requires a tight control in cell communication. A small number of signal transduction pathways such as Notch, Ras/MAPK, Hedgehog, Wnt, TGF-E, and JAK/STAT control these cellular interactions. These pathways are highly conserved among species and are essential for coordinating cellular differentiation, proliferation and apoptosis during development. Hence, these pathways are crucial for the diverse arrays of tissue (49). Our focus is to study Notch and Hedgehog signalling pathways. 1.1 The Notch signalling pathway 1.1.1 The discovery of Notch pathway In 1914 John S. Dexter described the first Notch mutant in D. melanogaster melanogaster. The Notch signalling pathway was named after the X-linked dominant D.melanogaster genetic mutants. These mutants exhibit irregular notches of missing tissue at the tips of the fly wing blades (130). However, the role of Notch in development was not appreciated until the 1940s, when the complete loss of Notch gene was found to cause lethal hyperplasia (165, 166). Further studies demonstrated that Notch deletion generates a "neurogenic" phenotype in cells destined to become epidermis. (5, 48). Notch lacks DNA binding domain, and in the nucleus heterodimerizes with the DNA binding protein CSL (203). Other studies demonstrate that Notch signalling is involved in many cellular processes of D.melanogaster and vertebrates, such as apoptosis, cell proliferation, differentiation and lineage decisions during embryonic development, as well as homeostasis of adult self-renewing organs (20). Finally, missregulation of Notch signalling has been associated with cancer (30, 56, 194, 202). 1.1.2 RBP-JN transcription factor RBP-JN (RBP-J) was first isolated as a 60 kDa DNA-binding protein from murine Bcell progenitors (61). RBP-J is part of the CSL family of protein and is highly conserved in a wide variety of organisms. In humans RBP-J is known as CBF-1, in Mammals D. melanogaster C. elegans Xenopus Receptor Notch-1-4 Notch Lin-12 X-Notch Glp-1 Ligand Delta-like 1,3 & 4 Delta Lag-2 X-Delta 1& 2 Jagged 1-2 Serrate Apx-1 X-Serrate-1 Arg-1 Dsl-1-7 Figure 1.1. Schematic representation of the Notch receptor. The extracellular domain consists of 36 EGF-like repeats (green) and 3 cysteine-rich repeats region (N/L) (blue). The intracellular domain consists of RAM domain (white), seven ankyrin repeats (orange), a glutamine OPA domain (grey), a proline, glutamine, serine and threonine rich (PEST) domain (black). (TACE), in X.laevis as Kuzbanian, and in C. elegans metalloproteinases are known as SUP-17 (Kuzbanian) and ADM-4 (TACE) (Figure 1.3) (20, 49, 101, 156). Finally, Notch is released into the intracellular space by the J-secretase enzymatic complex (S3) (Figure 1.3). J-secretase complex contains in D. melanogaster Presenilin, Nicastrin, APH-1, PEN-2, in C. elegans SEL-12, APH-1, APH-2, PEN-2, and in mammals Presenilin 1 and 2, Nicastrin, APH-1a-c, PEN-2 (101) 1.1.5 Function of the Notch signalling pathway Notch receptors and ligands are involved in the regulation of many biological functions, such as apoptosis, cell proliferation, differentiation and lineage decisions during embryonic development, and homeostasis of adult self-renewing organs (20). The Notch mechanisms of action in these cellular processes can be divided into four categories: lateral inhibition, lateral induction, boundary formation and asymmetric cell division and are summarized in figure 1.5. 1.1.8 RITA (RBP-J interacting and tubulin associated) Human RITA (RBP-J interacting and tubulin associated) was initially identified as an RBP-J interacting protein in a Yeast Two Hybrid screen. RITA is a 36 kDa protein, it has no significant homologies to any other protein and is highly conserved among species. RITA mediates the nuclear export of RBP-J to D-tubulin through a rapid nucleo-cytoplasmic shuttling. In X.laevis over-expression of RITA represses the transcriptional activation of notch target genes (208). SHH signalling can act in an autocrine fashion, affecting the cells in which it is produced, or act in a paracrine way, affecting the surrounding cells. The paracrine secretion of cholesterol modified SHH ligands is mediated by Dispatched1 protein (Disp1) (23, 86) (Figure 1.8_2). Disp1 is highly conserved in D.melanogaster, zebrafish, and mice remarking the importance of this mechanism (23, 90, 139, 206). Mice have two Disp homologs, Disp1 and Disp2, but only Disp1 is involved in the Hh signalling pathway (123). Once out of the cell, mammalian Hh ligands (Sonic, Indian and Desert) initiate signalling in the target cells by binding to the 12-pass
The Adaptor-associated Kinase 1, AAK1, Is a Positive Regulator of the Notch Pathway
Journal of Biological Chemistry, 2011
The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent ␥-secretase activity. ␥-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the ␥-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.