Cryogenic Ion Spectroscopy of a Singly Protonated Peptide DYYVVR: Locating Phosphorylation Sites of a Kinase Domain

2020 ◽  
Vol 11 (17) ◽  
pp. 7103-7108
Author(s):  
Jang Han Kwon ◽  
Min Ji Lee ◽  
Gyeongok Song ◽  
Kazuya Tsuruta ◽  
Shun-ichi Ishiuchi ◽  
...  
Keyword(s):  
Protonated Peptide ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Ion Spectroscopy

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.

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 Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Katja Hrovat Arnež ◽  
Michaela Kindlova ◽  
Nilesh J. Bokil ◽  
James M. Murphy ◽  
Matthew J. Sweet ◽  
...  
Keyword(s):  
Cell Death ◽  
Human Monocyte ◽  
Kinase Domain ◽  
Terminal Region ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Mixed Lineage Kinase

We show that mixed lineage kinase domain-like (MLKL) isoform 2, which lacks the pseudokinase domain and activation loop phosphorylation sites, is a more potent activator of cell death compared with MLKL isoform 1. Both MLKL isoforms are expressed in human monocyte-derived macrophages.

scholarly journals Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase domain-Like (MLKL) Interaction in Necroptotic Signaling

2013 ◽  
Vol 288 (23) ◽  
pp. 16247-16261 ◽  
Author(s):  
Wanze Chen ◽  
Zhenru Zhou ◽  
Lisheng Li ◽  
Chuan-Qi Zhong ◽  
Xinru Zheng ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Flanking Sequence ◽  
Interacting Protein ◽  
Mixed Lineage Kinase ◽  
Signaling Complex ◽  
Evolutionarily Conserved ◽  
Mouse Cells ◽  
Human And Mouse

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

scholarly journals The role of (auto)-phosphorylation in the complex activation mechanism of LRRK2

2018 ◽  
Vol 399 (7) ◽  
pp. 643-647 ◽  
Author(s):  
Panagiotis S. Athanasopoulos ◽  
Rolf Heumann ◽  
Arjan Kortholt
Keyword(s):  
Parkinson’S Disease ◽  
Late Onset ◽  
Kinase Domain ◽  
Activation Mechanism ◽  
Phosphorylation Sites ◽  
Gtpase Activity ◽  
Leucine Rich Repeat ◽  
Large Protein ◽  
N Terminus

AbstractMutations in human leucine-rich-repeat kinase 2 (LRRK2) have been found to be the most frequent cause of late-onset Parkinson’s Disease (PD). LRRK2 is a large protein with two enzymatic domains, a GTPase and a kinase domain. A cluster of (auto)-phosphorylation sites within the N-terminus of LRRK2 have been shown to be crucial for the localization of LRRK2 and is important for PD pathogenesis. In addition, phosphorylation of sites within the G-domain of the protein affect GTPase activity. Here we discuss the role of these (auto)-phosphorylation sites of LRRK2 and their regulation by phosphatases and upstream kinases.

scholarly journals Impact of Type II LRRK2 inhibitors on signalling and mitophagy

2021 ◽  
Author(s):  
Anna Tasegian ◽  
Francois Singh ◽  
Ian G Ganley ◽  
Alastair D Reith ◽  
Dario R Alessi
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Type I ◽  
Phosphorylation Sites ◽  
Type Ii ◽  
Closed Conformation ◽  
Lrrk2 Kinase ◽  
Target Effects ◽  
Physiological Substrates

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

Identification of Novel Phosphorylation Sites and Kinetics in Flt3 Receptor Wt, ITD and D835Y

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5395-5395
Author(s):  
Elena Razumovskaya ◽  
Kristina Masson ◽  
Rasheed Khan ◽  
Susanne Bengtsson ◽  
Lars Ronnstrand
Keyword(s):  
Tyrosine Kinase ◽  
Gene Mutations ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Wild Type ◽  
Over Expression ◽  
Juxtamembrane Region ◽  
Biological Responses

Abstract FMS-like tyrosine kinase-3 (Flt3) is a receptor tyrosine kinase, which is normally expressed in hematopoietic progenitor cells. It has been implicated as a major cause of transformation in acute myeloid leukemia (AML). There are two types of Flt3 gene mutations have been identified in AML: duplication of amino acids in the juxtamembrane region- Internal Tandem Duplication (ITD) and an activation loop point-mutation of D835 in kinase domain. These mutations cause constitutive activation or over expression of Flt3 receptor and therefore lead to alteration in signal transduction. These alterations occur in approximately 30% of AML patients. High occurrence of these mutations in the Flt3 receptor in AML patients makes it one of the most interesting therapeutic targets. In this study we have identified three novel in vivo phosphorylation sites of Flt3 receptor and further compared the activity of phosphorylation sites of Flt3 wild type, Flt3 ITD and D835Y mutations by using homemade phospho-specific antibodies directed against specific tyrosines. For this study murine hematopoietic Ba/F3 cells were stably transfected with wild-type Flt3, ITD and D835Y mutations. We have confirmed that the activation of the wild type Flt3 receptor is ligand dependent and response in a time dependent manner, but Flt3-ITD and D835Y are constitutive active and ligand independent. Phosphorylated tyrosines 589, 591, 599, 726, 768, 793, 842, and 955 of Flt3 receptor were investigated and shown to be differentially activated in wild-type versus the mutated receptor. Using this data we can further study the mechanisms of signaling pathways of the Flt3 receptor that are involved in many biological responses and understand the mechanism by which Flt3 ITD and D835Y functions in pathological conditions.

scholarly journals The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation

1998 ◽  
Vol 333 (3) ◽  
pp. 631-636 ◽  
Author(s):  
Pilar GARCIA-PARAMIO ◽  
Yolanda CABRERIZO ◽  
Frederic BORNANCIN ◽  
Peter J. PARKER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Inhibitory Protein ◽  
Kinase C ◽  
Broad Specificity

Dominant negative properties are conferred on protein kinase (PK) Cα by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCζ mutants (atypical isotype) inhibited both PKCα (classical isotype) and PKCε (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCα, consistent with the expected mode of action. In the case of the PKCα mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.

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.

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