Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase α

2005 ◽  
Vol 336 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Anna E. Szafranska ◽  
Xuemei Luo ◽  
Kevin N. Dalby
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Tryptic Peptide ◽  
Mitogen Activated Protein Kinase ◽  
Peptide Analysis ◽  
Mitogen Activated Protein Kinases ◽  
In Vitro Activation ◽  
Mitogen Activated Protein

scholarly journals The major phosphorylation site of the NADPH oxidase component p67phox is Thr233

1999 ◽  
Vol 338 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Louisa V. FORBES ◽  
Oanh TRUONG ◽  
Frans B. WIENTJES ◽  
Stephen J. MOSS ◽  
Anthony W. SEGAL
Keyword(s):  
Protein Kinase ◽  
Cyanogen Bromide ◽  
Tryptic Peptide ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Rich Domain ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping ◽  
Major Phosphorylation Site

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human

1999 ◽  
Vol 79 (1) ◽  
pp. 143-180 ◽  
Author(s):  
CHRISTIAN WIDMANN ◽  
SPENCER GIBSON ◽  
MATTHEW B. JARPE ◽  
GARY L. JOHNSON
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Mammalian Cells ◽  
Mitogen Activated Protein Kinase ◽  
Cell Physiology ◽  
Mitogen Activated Protein Kinases ◽  
Mapk Kinase ◽  
Mapk Pathways ◽  
Mitogen Activated Protein ◽  
Sequential Activation

Widmann, Christian, Spencer Gibson, Matthew B. Jarpe, and Gary L. Johnson. Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human. Physiol. Rev. 79: 143–180, 1999. — Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. Mitogen-activated protein kinases are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK. Currently, there have been 14 MKKK, 7 MKK, and 12 MAPK identified in mammalian cells. The mammalian MAPK can be subdivided into five families: MAPKerk1/2, MAPKp38, MAPKjnk, MAPKerk3/4, and MAPKerk5. Each MAPK family has distinct biological functions. In Saccharomyces cerevisiae, there are five MAPK pathways involved in mating, cell wall remodelling, nutrient deprivation, and responses to stress stimuli such as osmolarity changes. Component members of the yeast pathways have conserved counterparts in mammalian cells. The number of different MKKK in MAPK modules allows for the diversity of inputs capable of activating MAPK pathways. In this review, we define all known MAPK module kinases from yeast to humans, what is known about their regulation, defined MAPK substrates, and the function of MAPK in cell physiology.

scholarly journals Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells.

1993 ◽  
Vol 4 (8) ◽  
pp. 781-790 ◽  
Author(s):  
E K Shibuya ◽  
J V Ruderman
Keyword(s):  
Protein Kinases ◽  
Somatic Cells ◽  
Mapk Signaling ◽  
Mapk Activation ◽  
Free System ◽  
Mitogen Activated Protein Kinases ◽  
Activation Pathway ◽  
In Vitro Activation ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are rapidly and transiently activated when both quiescent Go-arrested cells and G2-arrested oocytes are stimulated to reenter the cell cycle. We previously developed a cell-free system from lysates of quiescent Xenopus oocytes that responds to oncogenic H-ras protein by activating a MAPK, p42MAPK. Here, we show that the oncogenic protein kinase mos is also a potent activator of p42MAPK in these lysates. Mos also induces p42MAPK activation in lysates of activated eggs taken at a time when neither mos nor p42MAPK is normally active, showing that the mos-responsive MAPK activation pathway persists beyond the stage where mos normally functions. Similarly, lysates of somatic cells (rabbit reticulocytes) also retain a mos-inducible MAPK activation pathway. The mos-induced activation of MAPKs in all three lysates leads to phosphorylation of the pp90rsk proteins, downstream targets of the MAPK signaling pathway in vivo. The in vitro activation of MAPKs by mos in cell-free systems derived from oocytes and somatic cells suggests that mos contributes to oncogenic transformation by inappropriately inducing the activation of MAPKs.

scholarly journals Regulation of proline-directed kinases and the trans-histone code H3K9me3/H4K20me3 during human myogenesis

2019 ◽  
Vol 294 (20) ◽  
pp. 8296-8308 ◽  
Author(s):  
Natarajan V. Bhanu ◽  
Simone Sidoli ◽  
Zuo-Fei Yuan ◽  
Rosalynn C. Molden ◽  
Benjamin A. Garcia
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Isotope Labeling ◽  
Proteome Analysis ◽  
Mitogen Activated Protein Kinase ◽  
System Level ◽  
Post Translational Modification ◽  
Mitogen Activated Protein ◽  
Myoblast Cell

We present a system-level analysis of proteome, phosphoproteome, and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling of amino acids in cell culture and nano-liqud chromatography-mass spectrometry/mass spectrometry, we found that phosphorylation motifs targeted by the kinases protein kinase C, cyclin-dependent kinase, and mitogen-activated protein kinase showed increased phosphorylation during myodifferentiation of LHCN-M2 human skeletal myoblast cell line. Drugs known to inhibit these kinases either promoted (PD0325901 and GW8510) or stalled (CHIR99021 and roscovitine) differentiation, resulting in myotube and myoblast phenotypes, respectively. The proteomes, especially the myogenic and chromatin-related proteins including histone methyltransferases, correlated with their phenotypes, leading us to quantify histone post-translational modifications and identify two gene-silencing marks, H3K9me3 and H4K20me3, with relative abundances changing in correlation with these phenotypes. ChIP–quantitative PCR demonstrated that H3K9me3 is erased from the gene loci of myogenic regulatory factors namely MYOD1, MYOG, and MYF5 in differentiating myotubes. Together, our work integrating histone post-translational modification, phosphoproteomics, and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during myodifferentiation in vitro.

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