Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

scholarly journals Structural differences between repressed and derepressed forms of p60c-src.

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656 ◽  
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

scholarly journals The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy.

1996 ◽  
Vol 134 (6) ◽  
pp. 1441-1453 ◽  
Author(s):  
W M Obermann ◽  
M Gautel ◽  
F Steiner ◽  
P F van der Ven ◽  
K Weber ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Terminal Region ◽  
M Protein ◽  
Amino Terminal ◽  
Thick Filaments ◽  
Carboxy Terminal Region ◽  
Oriented Parallel ◽  
Carboxy Terminal

The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.

scholarly journals SEL-5, A Serine/Threonine Kinase That Facilitates lin-12 Activity in Caenorhabditis elegans

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1641-1654 ◽  
Author(s):  
Hanna Fares ◽  
Iva Greenwald
Keyword(s):  
Cell Fate ◽  
Point Mutations ◽  
Kinase Domain ◽  
Terminal Region ◽  
Serine Threonine Kinase ◽  
Intracellular Domain ◽  
Cell Fate Decisions ◽  
Threonine Kinase ◽  
Amino Terminal ◽  
Fate Decision

Abstract Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.

scholarly journals Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

2009 ◽  
Vol 29 (12) ◽  
pp. 3367-3378 ◽  
Author(s):  
Scott A. Robertson ◽  
Rositsa I. Koleva ◽  
Lawrence S. Argetsinger ◽  
Christin Carter-Su ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Feedback Inhibition ◽  
Dominant Role ◽  
Kinase Domain ◽  
Cytokine Signaling ◽  
Tyrosine Kinase Domain ◽  
Phosphorylation Sites ◽  
Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively

1993 ◽  
Vol 13 (1) ◽  
pp. 133-143
Author(s):  
M Valius ◽  
C Bazenet ◽  
A Kazlauskas
Keyword(s):  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphates ◽  
Platelet Derived Growth Factor ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Mitogenic Response ◽  
Stable Association ◽  
64 Kda Protein ◽  
Carboxy Terminal

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

scholarly journals Variable expression of immunoreactive surface proteins of Propionibacterium acnes

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3667-3681 ◽  
Author(s):  
Michael J. Lodes ◽  
Heather Secrist ◽  
Darin R. Benson ◽  
Shyian Jen ◽  
Kurt D. Shanebeck ◽  
...  
Keyword(s):  
T Cell ◽  
Propionibacterium Acnes ◽  
Cell Epitope ◽  
Terminal Region ◽  
Clinical Isolates ◽  
Carboxy Terminus ◽  
Dermatan Sulphate ◽  
Variable Expression ◽  
Lpxtg Motif ◽  
Amino Terminal

Despite accumulating data implicating Propionibacterium acnes in a variety of diseases, its precise role in infection remains to be determined. P. acnes antigen-specific CD4+ T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens from P. acnes expression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein of Streptococcus equi and have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of the Corynebacterium diphtheriae htaA gene from the hmu ABC transport locus, although only one of these (PA-21693) is encoded within an hmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4+ T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role of P. acnes in inflammatory acne and other diseases.

CARBOHYDRATE PREVENTS LOSS OF LARGE VON WILLEBRAND FACTOR MULTIMERS BY PROTECTING AGAINST AMINO TERMINAL PROTEOLYTIC CLEAVAGE

1987 ◽  
Author(s):  
A B Federici ◽  
S D Berkowitz
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Enzymatic Digestion ◽  
Terminal Region ◽  
Cleavage Sites ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

We have previously shown that carbohydrate (CHO) protects von Willebrand factor (vWF) from proteolytic degradation. We have now shown that removal of CHO from the vWF subunit exposes additional cleavage sites in the amino terminal region and that cleavages in this region are associated with loss of large multimers. We examined and compared the extent of large multimer loss with sites of subunit cleavage of native and GHO-modified vWF after treatment with plasmin, chymotrypsin, and trypsin. Highly purified vWF was treated with neuraminidase and β-galactosidase in the presence of proteinase inhibitors to remove 90-95% of the sialic acid and 45-50% of the D-galactose without loss of large multimers or diminution of the ristocetin cofactor activity. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Multimeric analysis revealed an increasingly greater loss of large multimers when native vWF was digested with plasmin, chymotrypsin, and trypsin, respectively. Large multimer loss was more extensive with each enzyme after CHO-modification of vWF. On subunit analysis, plasmin, chymotrypsin, and trypsin were shown to produce both amino and carboxy terminal fragments. The number, location, and relative quantities of carboxy terminal fragments produced by these enzymes were unchanged after CHO modification. However, digestion of the amino terminal region was considerably more extensive as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular weight species. Thus, enzymatic digestion of vWF after removal of carbohydrate produced new cleavages in the amino terminal region but did not alter the location or extent of carboxy terminal cleavages. Therefore, the greater loss of large multimers that occurs after CHO modification is likely to be the result of cleavages in the amino terminal region of the molecule. It appears that by protecting the vWF subunit against amino terminal cleavage, carbohydrate inhibits the loss of large multimers.

Melittin: a Membrane-active Peptide with Diverse Functions

2007 ◽  
Vol 27 (4-5) ◽  
pp. 189-223 ◽  
Author(s):  
H. Raghuraman ◽  
Amitabha Chattopadhyay
Keyword(s):  
Protein Interactions ◽  
Terminal Region ◽  
Membrane Properties ◽  
Suitable Model ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Amino Terminal ◽  
Cellular Processes ◽  
Lipid Protein Interactions ◽  
Carboxy Terminal

Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property of melittin has resulted in melittin being used as a suitable model peptide for monitoring lipid–protein interactions in membranes. In this review, the solution and membrane properties of melittin are highlighted, with an emphasis on melittin–membrane interaction using biophysical approaches. The recent applications of melittin in various cellular processes are discussed.

scholarly journals Crystal structure of the WD40 domain dimer of LRRK2

2019 ◽  
Vol 116 (5) ◽  
pp. 1579-1584 ◽  
Author(s):  
Pengfei Zhang ◽  
Ying Fan ◽  
Heng Ru ◽  
Li Wang ◽  
Venkat Giri Magupalli ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Human Genetics ◽  
Kinase Domain ◽  
Rab Gtpases ◽  
Kinase Activation ◽  
Surface Patches ◽  
Wd40 Domain ◽  
Lrrk2 Kinase

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein with both a Ras of complex (ROC) domain and a kinase domain (KD) and, therefore, exhibits both GTPase and kinase activities. Human genetics studies have linked LRRK2 as a major genetic contributor to familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder that inflicts millions worldwide. The C-terminal region of LRRK2 is a Trp-Asp-40 (WD40) domain with poorly defined biological functions but has been implicated in microtubule interaction. Here, we present the crystal structure of the WD40 domain of human LRRK2 at 2.6-Å resolution, which reveals a seven-bladed WD40 fold. The structure displays a dimeric assembly in the crystal, which we further confirm by measurements in solution. We find that structure-based and PD-associated disease mutations in the WD40 domain including the common G2385R polymorphism mainly compromise dimer formation. Assessment of full-length LRRK2 kinase activity by measuring phosphorylation of Rab10, a member of the family of Rab GTPases known to be important kinase substrates of LRRK2, shows enhancement of kinase activity by several dimerization-defective mutants including G2385R, although dimerization impairment does not always result in kinase activation. Furthermore, mapping of phylogenetically conserved residues onto the WD40 domain structure reveals surface patches that may be important for additional functions of LRRK2. Collectively, our analyses provide insights for understanding the structures and functions of LRRK2 and suggest the potential utility of LRRK2 kinase inhibitors in treating PD patients with WD40 domain mutations.

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