scholarly journals A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas

2007 ◽  
Vol 176 (6) ◽  
pp. 819-829 ◽  
Author(s):  
Lai-Wa Tam ◽  
Nedra F. Wilson ◽  
Paul A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Cell Fractionation ◽  
Cyclin Dependent Kinase ◽  
Yeast Two Hybrid ◽  
Flagellar Assembly ◽  
Two Hybrid

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.

Identification of the TIMP-2 Binding Site on the Gelatinase A Hemopexin C-Domain by Site-Directed Mutagenesis and the Yeast Two-Hybrid System

1999 ◽  
Vol 878 (1 INHIBITION OF) ◽  
pp. 747-753 ◽  
Author(s):  
CHRISTOPHER M. OVERALL ◽  
ANGELA E. KING ◽  
HEATHER F. BIGG ◽  
ANGUS McQUIBBAN ◽  
JULIET ATHERSTONE ◽  
...  
Keyword(s):  
Binding Site ◽  
Hybrid System ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gelatinase A ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Phosphorylation of BIK1 is critical for interaction of downstream signaling components

2021 ◽  
Author(s):  
Jae-Han Choi ◽  
Eun-Seok Oh ◽  
Man-Ho Oh
Keyword(s):  
Growth And Development ◽  
Kinase Activity ◽  
Higher Plants ◽  
Site Directed Mutagenesis ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Downstream Signaling ◽  
Signaling Components ◽  
Two Hybrid

Abstract Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. Interestingly, Arabidopsis thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas Brassica rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a yeast two-hybrid (Y2H) system. We subsequently employed bimolecular fluorescence complementation (BiFC) to confirm interactions between BIK1 and candidates in Nicotiana benthamiana, and found that only BrBIK1 bound the three proteins tested. We selected three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A368T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable with AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. Interestingly, BiFC results showed that BIK1 mutations restored kinase activity but not binding to RGP2, PATL2, or PP7 proteins. Our results suggest that phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.

scholarly journals Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses

2000 ◽  
Vol 149 (7) ◽  
pp. 1419-1432 ◽  
Author(s):  
Ute Schaeper ◽  
Niels H. Gehring ◽  
Klaus P. Fuchs ◽  
Martin Sachs ◽  
Bettina Kempkes ◽  
...  
Keyword(s):  
Binding Site ◽  
Mdck Cells ◽  
Mapk Pathway ◽  
Biological Response ◽  
Adaptor Protein ◽  
Branching Morphogenesis ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Interaction Sites ◽  
Two Hybrid

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met–specific branching morphogenesis. It associates directly with c-Met via the c-Met–binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met–binding site is localized to a 13–amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met–binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1–specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Cyclin I and p35 determine the subcellular distribution of Cdk5

2015 ◽  
Vol 308 (4) ◽  
pp. C339-C347 ◽  
Author(s):  
Henning Hagmann ◽  
Yoshinori Taniguchi ◽  
Jeffrey W. Pippin ◽  
Hans-Michael Kauerz ◽  
Thomas Benzing ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Cell Biology ◽  
Kinase Activity ◽  
Brain Slices ◽  
Axonal Guidance ◽  
Cell Fractionation ◽  
Cyclin Dependent Kinase ◽  
Immunofluorescence Labeling ◽  
Cellular Compartments ◽  
Cyclin I

The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals Characterization of iron-binding motifs in Candida albicans high-affinity iron permease CaFtr1p by site-directed mutagenesis

2002 ◽  
Vol 368 (2) ◽  
pp. 641-647 ◽  
Author(s):  
Hao-Ming FANG ◽  
Yue WANG
Keyword(s):  
Amino Acids ◽  
Candida Albicans ◽  
Iron Uptake ◽  
Iron Transport ◽  
Site Directed Mutagenesis ◽  
Detectable Effect ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Iron Binding ◽  
Direct Binding

A peptide motif Glu-Xaa-Xaa-Glu has been implicated in direct binding of ferric iron in several proteins involved in iron transport, sensing or storage. However, it is not known whether the motif alone is sufficient for iron binding and whether functional replacement of the conserved residues by other amino acids with similar properties is possible. We previously identified a Candida albicans iron permease, CaFtr1p, which contains five Glu-Xaa-Xaa-Glu motifs [Ramanan and Wang (2000) Science 288, 1062—1065]. In this study, we investigated the role of each of these motifs in iron uptake by site-directed mutagenesis. Substitution of Ala for any one of the two Glu residues in Glu-Gly-Leu-Glu158—161 abolished iron-uptake activity, while the same substitution in any of the other four motifs had little effect, indicating that only the motif at position 158—161 is required for iron transport. We then evaluated the importance of each of the residues within and immediately adjacent to this motif in iron uptake. The permease remained active when any one of the Glu residues was replaced by Asp, while it became inactive when both were replaced. We also found that the amino acid immediately in front of Glu-Gly-Leu-Glu158—161 must be either Arg or Lys. In addition, substitution of any of the two residues in the middle with several structurally distinct amino acids had no detectable effect on iron uptake. Here we propose to extend the iron-binding motif to Arg/Lys-Glu/Asp-Xaa-Xaa-Glu or Arg/Lys-Glu-Xaa-Xaa-Glu/Asp, which may serve as a guide for the identification of potential iron-binding sites in proteins.

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