Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

A new mild experimental approach for isolation of peptide membrane receptors and subsequent analysis of post-translational modifications is described. Endothelin receptors A and B were isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide endothelin coupled to a protected (dA)-30-mer. This allowed a one-step isolation of the receptor from oligo(dT)-cellulose via variation solely of salt concentration. The identity of the receptor was confirmed by direct amino acid sequencing of electroblotted samples or by using antibodies against ET A and ET B receptors. Although it is clear that post-translational modifications of G-protein-coupled receptors are intimately involved in the physiological function of these signal transduction systems, there is as yet relatively little direct evidence for the specific modifications and the relationship of the different modifications to signal transduction processes. These types of processes are not easily amenable to analysis by genomic methods, i.e. there is a need for efficient methods to directly analyze these processes at the proteome level. We report here new, efficient methods for rapid isolation of the endothelin B receptor and for highly sensitive analysis of its post-translational modifications via mass spectrometry.Endothelin, the strongest vasoconstrictor yet known, is a 21-amino acid peptide with physiological effects on cellular development, differentiation, vasoconstriction, and mitogenesis (1, 2). There are 3 different endothelin isoforms, ET-1, 1 ET-2, and ET-3 with different affinity for the two different endothelin receptor subtypes, A and B (3-6). Both receptors are members of the G-protein-coupled receptor superfamily (3-6, 7). The consensus ET receptor topology includes three extracellular domains, three intracellular loops, and a cytoplasmic COOH-terminal tail, separated by seven hydrophobic helical regions thought to span the lipid bilayer. In addition it has been presumed that ET receptors are post-translationally modified by glycosylation of the NH 2 terminus and by phosphorylation and palmitoylation of the cytoplasmic surface. Based on homology with other G-protein-coupled receptors (8 -10), there has been speculation regarding possible structures, functional regions, and sites of post-translational modifications for endothelin receptors (11-16). However, as yet there is very little direct evidence for attributes such as the sites and the roles of glycosylation, palmitoylation, and phosphorylation, the location of the endothelin-binding site and the basis for discrimination among the three different endothelin isoforms.A role of endothelin in disease has recently been demonstrated by the finding that mutated ET B receptor is associated with Hirschsprung's disease (17-21). In addition, mice lacking the ET-1 gene display severe malformation of large blood vessels, stressing the importance of endothelin during development (22). Endothelin has been shown to be a mitogenic agonist in different cell types (23, 24). The signaling pathway by ...

Section: Isolation Of Et B Receptorsupporting

confidence: 75%

Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

Roos¹,

Šoškić²,

Poznanović³

et al. 1998

“…Indeed, Ca 2ϩ may have a widespread and fundamental role in suppressing myosin I motile activity, because the vertebrate myosin I isoforms are inhibited at elevated levels of Ca 2ϩ through a Ca 2ϩ -dependent dissociation of the calmodulin light chains (48). Recently, the Acanthamoeba MIHCK has been reported to contain a putative Cdc42/Rac binding site (39), suggesting that its activity is likely to be regulated in some manner by these small GTPases. Thus, the framework for a conserved set of intracellular signals that are involved in the control of myosin I-driven motile events in lower eukaryotes is beginning to emerge.…”

Section: ؉mentioning

confidence: 99%

“…The finding that activation of Dictyostelium MIHCK results from the phosphorylation of a site close to the amino terminus is somewhat unexpected, because the activation of PAK (36,37), Ste20p (38), and the isolated Acanthamoeba MIHCK catalytic domain (39) is closely tied to autophosphorylation of a Thr in a region of the catalytic domain termed the "activation segment" (reviewed in Ref. 40).…”

Section: ؉mentioning

confidence: 99%

Regulation of the p21-activated Kinase-relatedDictyostelium Myosin I Heavy Chain Kinase by Autophosphorylation, Acidic Phospholipids, and Ca2+-Calmodulin

Lee

Mahasneh

Côté

1998

“…The identity of the peptides was confirmed by electrospray ion trap mass spectrometry of the unseparated peptide mixture with subsequent MS/MS analysis of selected fragments of interest. It is known that in electrospray ion trap mass spectrometry the phosphorylated peptides partially lose the H 3 PO 4 moiety in the mass spectrometer, thus producing a pair of peaks separated by a mass difference of 98 Da (18). In accordance with this, we observed that phosphopeptides 6 -7, 26 -31, and 28 -31 could be identified by a pair of masses 80 Da higher (the mass of H 3 PO 4 minus H 2 O) and 18 Da lower than expected based on the amino acid sequence.…”

Section: Phosphorylation/palmitoylation Of Bradykinin B 2 Receptormentioning

confidence: 99%

Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

Šoškić¹,

Nyakatura²,

Roos³

et al. 1999

Rat bradykinin B 2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B 2 receptor was isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA) 30 -mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B 2 receptor showed phosphorylation at Bradykinin, a member of the kinin family (1), is a nonapeptide with diverse biological activities ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3). Bradykinin mediates its physiological effects by binding to and activation of the bradykinin B 2 receptor. Molecular cloning has revealed the primary structure of the B 2 receptor (4) and classified it as a member of the G protein-coupled receptor superfamily. The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is still rather little direct evidence at the protein level for attributes such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B 2 or other G protein-coupled receptors. Indeed phosphorylation sites for few G protein-coupled receptors have been mapped to date (10 -12), and most of these sites reflect the in vitro phosphorylation of the isolated receptor.We report here on the isolation of rat B 2 receptor from transfected Chinese hamster ovary (CHO) cells using oligo(dA) covalently linked to bradykinin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out under very mild conditions using oligo(dT) columns analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically separated receptor, subsequent peptide mass fingerprinting by matrix-assisted laser desorption...