Identification of new in vivo phosphosites on lamin Dm - the evidence of heterogeneity of phosphorylation sites in differentDrosophilatissues

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

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Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.00067-09 ◽

2009 ◽

Vol 8 (7) ◽

pp. 922-932 ◽

Cited By ~ 37

Author(s):

Jens Boesger ◽

Volker Wagner ◽

Wolfram Weisheit ◽

Maria Mittag

Keyword(s):

Chlamydomonas Reinhardtii ◽

Protein Phosphorylation ◽

Proteome Analysis ◽

Physiological Status ◽

Phosphorylation Sites ◽

Cell Organelles ◽

Flagellar Proteins ◽

Reversible Protein Phosphorylation ◽

Cilia And Flagella

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

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High-accuracy identification and bioinformatic analysis of in vivo protein phosphorylation sites in yeast

PROTEOMICS ◽

10.1002/pmic.200900144 ◽

2009 ◽

Vol 9 (20) ◽

pp. 4642-4652 ◽

Cited By ~ 90

Author(s):

Florian Gnad ◽

Lyris M. F. de Godoy ◽

Jürgen Cox ◽

Nadin Neuhauser ◽

Shubin Ren ◽

...

Keyword(s):

Protein Phosphorylation ◽

Bioinformatic Analysis ◽

High Accuracy ◽

Phosphorylation Sites

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Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo.

The EMBO Journal ◽

10.1002/j.1460-2075.1992.tb05182.x ◽

1992 ◽

Vol 11 (4) ◽

pp. 1373-1382 ◽

Cited By ~ 181

Author(s):

A. Kashishian ◽

A. Kazlauskas ◽

J.A. Cooper

Keyword(s):

Pi3 Kinase ◽

Phosphorylation Sites ◽

Pdgf Receptor

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Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3227-3237.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3227-3237 ◽

Cited By ~ 26

Author(s):

Kazuhiro Maki ◽

Honoka Arai ◽

Kazuo Waga ◽

Ko Sasaki ◽

Fumihiko Nakamura ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Effect ◽

Phosphorylation Sites ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

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Identification of Two Src Recruitment Sites in the Juxtamembrane Region of Flt3 with Opposing Effects on Flt3-Ligand-Induced Signaling.

Blood ◽

10.1182/blood.v106.11.2289.2289 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2289-2289

Author(s):

Lars Ronnstrand ◽

Elke Heiss ◽

Christina Sundberg ◽

Kristina Masson ◽

Malin Pedersen ◽

...

Keyword(s):

Signal Transduction ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Src Family Kinases ◽

Phosphorylation Sites ◽

Flt3 Ligand ◽

Erk Activation ◽

Juxtamembrane Region ◽

Signal Relay

Abstract Early signal relay steps upon ligand-binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3-autophosphorylation and subsequent docking partners, are mainly unresolved. Here we demonstrate for the first time identification of ligand-induced in vivo phosphorylation sites in Flt3. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3, Y589F-Flt3 and Y599F-Flt3 expressing 32D cells. Compared to WT-Flt3-32D cells, 32D-Y589F-Flt3 showed upon ligand-stimulation enhanced Erk activation as well as proliferation/survival whereas 32D-Y599F-Flt3 cells displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for multiple signal relay molecules including Src family kinases. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered Src activation, impaired phosphorylation of the adapter molecules Cbl and ShcA and deficient receptor ubiquitination and degradation. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. pY599 was additionally found to interact with the protein tyrosine phosphatase Shp2. As Y599F-Flt3-32D lacked ligand-induced Shp2 phosphorylation and since silencing of Shp2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of Shp2 to pY599 contributes to FL-mediated Erk activation and proliferation. To summarize, our work presents novel insights in Flt3-mediated signal transduction. We have identified the in vivo autophosphorylation sites of the juxtamembrane region of Flt3, revealed Src family kinases and Shp2 as binding partners of pY589 and/or pY599, respectively, as well as their potential impact on FL-mediated signaling in Flt3-32D cells. Future work will now focus on elucidation of additional and possibly novel interaction partners of the found phosphorylation sites by employing an unbiased proteomics approach. With this gained knowledge it will be of interest to see whether ITDs differing in the nature of the duplicated tyrosines also confer distinct signaling behavior. If so, these tyrosines might serve as a diagnostic marker and point towards a successful combinatorial therapy consisting of a receptor tyrosine kinase inhibitor and an inhibitor for the specifically affected signal transduction pathway.

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Identification of Tyrosine Residues of Importance for Survival Signaling through the Scaffolding Protein Gab2 in Both Wild-Type FLT3 and the FLT3-ITD.

Blood ◽

10.1182/blood.v110.11.1622.1622 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1622-1622

Author(s):

Kristina Masson ◽

Tao Liu ◽

Jianmin Sun ◽

Lars Ronnstrand

Keyword(s):

Scaffolding Protein ◽

Phosphorylation Sites ◽

Wild Type ◽

Internal Tandem Duplication ◽

Tyrosine Residues ◽

Downstream Signaling ◽

Juxtamembrane Region ◽

Future Potential ◽

Survival Signaling

Abstract The receptor tyrosine kinase FLT3 is normally expressed in hematopoietic progenitor cells and has been implicated as a major cause of transformation in acute myeloid leukemia, where it in approximately 30% of cases is mutated and constitutively active. This is in most cases due to duplication of a DNA sequence coding for amino acids in the juxtamembrane region of FLT3, commonly referred to as ITD (Internal Tandem Duplication). In this study we have identified several novel in vivo tyrosine phosphorylation sites that are phosphorylated in wild-type FLT3 upon ligand stimulation and that are constitutively phosphorylated in the FLT3-ITD. We were able to demonstrate that these phosphorylation sites are critical for full phosphorylation of the scaffolding protein Gab2 both in wild-type FLT3 and FLT3-ITD. Y-to-F mutants of either wild-type FLT3 or FLT3-ITD, lacking these tyrosine residues, fail to phosphorylate Gab2 and demonstrate a considerable reduction in phosphorylation of Akt and Erk. Furthermore, FL-dependent survival and proliferation of wild-type FLT3 expressing Ba/F3 cells as well as FL-independent survival and proliferation of Ba/F3 cells transfected with FLT3-ITD was dramatically reduced by mutation of these tyrosine residues. In the case of the FLT3-ITD, this was shown to correlate with strongly reduced STAT5 phosphorylation. To verify the importance of Gab2 in FLT3-ITD signaling, we used siRNA technology to knock down the expression of Gab2 in the human AML cell line MV4-11 that is known to express FLT3-ITD. Knockdown of Gab2 expression led to a dramatic reduction in the phosphorylation of Akt, Erk and Stat5. To summarize, we have identified novel phosphorylation sites in FLT3 and how they link to downstream signaling of survival and proliferation. These findings not only reveal novel phosphorylation sites in FLT3 but also contribute to the understanding of the molecular mechanism by which FLT3-ITD functions in pathological conditions. Future studies are aiming at elucidating the mechanism by which Gab2 mediates phosphorylation and activation of STAT5, which could be a future potential target for therapy in AML with FLT3-ITD.

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