Biochemical characterization and localization of the dual specificity kinase CLK1

2000 ◽  
Vol 113 (18) ◽  
pp. 3241-3253 ◽  
Author(s):  
H.J. Menegay ◽  
M.P. Myers ◽  
F.M. Moeslein ◽  
G.E. Landreth
Keyword(s):  
Enzymatic Activity ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Splice Variants ◽  
Fold Increase ◽  
Small Subset ◽  
Tyrosine Phosphatases ◽  
Neuronal Populations ◽  
Dual Specificity ◽  
N Terminus

CLK1 was one of the first identified dual specificity kinases and is the founding member of the ‘LAMMER’ family of kinases. We have established the substrate site specificity of CLK1. We report here that truncation of the N terminus of CLK1 resulted in a dramatic increase in CLK1 enzymatic activity, indicating that the N terminus acts as a negative regulatory domain. The N-terminal truncation resulted in a 45-fold increase in V(max), suggesting that this domain does not contain a pseudo-substrate motif, but may act to conformationally constrain the catalytic activity of CLK1. Tyrosine phosphorylation has been proposed to be critical for CLK1 activity, however, CLK1 activity was unaffected by exposure to tyrosine phosphatases. Treatment of CLK1 with the serine/threonine specific phosphatase PP2A, resulted in a 2- to 6-fold increase in enzymatic activity. Incubation of CLK1 with tyrosine phosphatases in combination with PP2A abolished CLK1 activity. These data suggest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity. CLK1 activity is positively regulated by phosphorylation on either tyrosine residues or serine/threonine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of serine/threonine residues within the catalytic domain. CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and truncated splice forms are expressed at roughly equivalent levels in most tissues. The ratio of the two splice variants of CLK1 can be altered by treatment with cycloheximide. CLK1 protein expression is limited to a small subset of highly localized neuronal populations in the rat brain. Contrary to previous studies using overexpression systems, we show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.

scholarly journals Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7)

2016 ◽  
Vol 165 ◽  
pp. 7-17 ◽  
Author(s):  
Fan Meng ◽  
Hao Yang ◽  
Colin Jack ◽  
Huaqun Zhang ◽  
Abraham Moller ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Zinc Binding ◽  
Inhibition Studies

On mechanisms of colicin import: the outer membrane quandary

2018 ◽  
Vol 475 (23) ◽  
pp. 3903-3915 ◽  
Author(s):  
William A. Cramer ◽  
Onkar Sharma ◽  
S.D. Zakharov
Keyword(s):  
Outer Membrane ◽  
Catalytic Domain ◽  
Efflux Pump ◽  
Crystal Structure Analysis ◽  
Multidrug Efflux Pump ◽  
Terminal Domain ◽  
Colicin E1 ◽  
N Terminus ◽  
T Domain

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10−9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

scholarly journals Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

2018 ◽  
Vol 19 (10) ◽  
pp. 3099 ◽  
Author(s):  
Anna Malekkou ◽  
Maura Samarani ◽  
Anthi Drousiotou ◽  
Christina Votsi ◽  
Sandro Sonnino ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Autosomal Recessive ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Loss Of Function ◽  
Spastic Paraplegia ◽  
Spastic Ataxia ◽  
Autosomal Recessive Cerebellar Ataxia ◽  
Compensatory Effect

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

scholarly journals Structure of human dual-specificity phosphatase 7, a potential cancer drug target

2015 ◽  
Vol 71 (6) ◽  
pp. 650-656 ◽  
Author(s):  
George T. Lountos ◽  
Brian P. Austin ◽  
Joseph E. Tropea ◽  
David S. Waugh
Keyword(s):  
Catalytic Domain ◽  
Three Dimensional ◽  
Mitogen Activated Protein Kinase ◽  
Cancer Drug ◽  
Dual Specificity Phosphatase ◽  
Dual Specificity ◽  
Starting Point ◽  
Mitogen Activated Protein ◽  
Dual Specificity Phosphatases

Human dual-specificity phosphatase 7 (DUSP7/Pyst2) is a 320-residue protein that belongs to the mitogen-activated protein kinase phosphatase (MKP) subfamily of dual-specificity phosphatases. Although its precise biological function is still not fully understood, previous reports have demonstrated that DUSP7 is overexpressed in myeloid leukemia and other malignancies. Therefore, there is interest in developing DUSP7 inhibitors as potential therapeutic agents, especially for cancer. Here, the purification, crystallization and structure determination of the catalytic domain of DUSP7 (Ser141–Ser289/C232S) at 1.67 Å resolution are reported. The structure described here provides a starting point for structure-assisted inhibitor-design efforts and adds to the growing knowledge base of three-dimensional structures of the dual-specificity phosphatase family.

scholarly journals Phosphorylation of Serine 1106 in the Catalytic Domain of Topoisomerase IIα Regulates Enzymatic Activity and Drug Sensitivity

2003 ◽  
Vol 278 (15) ◽  
pp. 12696-12702 ◽  
Author(s):  
Kenichi Chikamori ◽  
Dale R. Grabowski ◽  
Michael Kinter ◽  
Belinda B. Willard ◽  
Satya Yadav ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Drug Sensitivity ◽  
Catalytic Domain ◽  
Topoisomerase Iiα

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

scholarly journals Biochemical characterization of selected plant species from Brazilian Savannas

2004 ◽  
Vol 47 (2) ◽  
pp. 253-259 ◽  
Author(s):  
Samantha Salomão Caramori ◽  
Claudinei Sousa Lima ◽  
Kátia Flávia Fernandes
Keyword(s):  
Enzymatic Activity ◽  
Plant Species ◽  
Biochemical Characterization ◽  
Hymenaea Courbaril ◽  
Annona Crassiflora

The aim of this work was to analyze and quantify the presence of antinutritional compounds such as lectins and trypsin-like inhibitors, polyphenols and tannins, and enzymatic activity of peroxidases and proteases in the seeds of Annona crassiflora Mart. (araticum), Hymenaea courbaril L. var. courbaril (jatobá), Plathymenia reticulata Benth. (vinhático), Zanthoxylum rhoifolium Lam. (maminha de porca), Apeiba tibourbou Aubl. (pau jangada), Salacia crassiflora Mart G. Don. (bacupari), and Sclerolobium paniculatum Vog. (carvoeiro). The results suggested that these plants could be used as new source of food.

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