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

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

Download Full-text

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00582.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. H2089-H2095 ◽

Cited By ~ 27

Author(s):

Brian B. Roman ◽

Paul H. Goldspink ◽

Elyse Spaite ◽

Dalia Urboniene ◽

Ron McKinney ◽

...

Keyword(s):

Troponin I ◽

Cardiac Contraction ◽

Phosphorylation Sites ◽

Developed Pressure ◽

Contraction And Relaxation ◽

Intracellular Targets ◽

Camp Levels

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.

Download Full-text

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.

Download Full-text

Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4131 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4131-4140 ◽

Cited By ~ 154

Author(s):

Christopher D. Kontos ◽

Thomas P. Stauffer ◽

Wen-Pin Yang ◽

John D. York ◽

Liwen Huang ◽

...

Keyword(s):

Fluorescent Protein ◽

Vascular Development ◽

Pi3 Kinase ◽

Pleckstrin Homology Domain ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Embryonic Vascular Development ◽

Phosphatidylinositol 3

ABSTRACT Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Nucleus ◽

10.4161/nucl.2.5.17864 ◽

2011 ◽

Vol 2 (5) ◽

pp. 478-488 ◽

Cited By ~ 8

Author(s):

Magdalena Zaremba-Czogalla ◽

Przemysław Gagat ◽

Katarzyna Kozioł ◽

Magda Dubińska-Magiera ◽

Jacek Sikora ◽

...

Keyword(s):

Phosphorylation Sites

Download Full-text