A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

2012 ◽  
Vol 427 (2) ◽  
pp. 127-129 ◽  
Author(s):  
Mieder A.T. Palm-Forster ◽  
Lennart Eschen-Lippold ◽  
Justin Lee
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Mitogen Activated Protein ◽  
Kinase Substrates

Phosphorylation of Grb10 by Mitogen-Activated Protein Kinase:  Identification of Ser150and Ser476of Human Grb10ζ as Major Phosphorylation Sites†

Biochemistry ◽  
2005 ◽  
Vol 44 (24) ◽  
pp. 8890-8897 ◽  
Author(s):  
Paul Langlais ◽  
Changhua Wang ◽  
Lily Q. Dong ◽  
Christopher A. Carroll ◽  
Susan T. Weintraub ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Mitogen Activated Protein

Postal code for a plant MAPK

2012 ◽  
Vol 446 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Brian E. Ellis
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Postal Code ◽  
Biochemical Journal ◽  
Mitogen Activated Protein ◽  
Cellular Signal ◽  
Efficient Route ◽  
Kinase Substrates

Plants contain hundreds of protein kinases that are believed to provide cellular signal transduction services, but the identities of the proteins they are targeting are largely unknown. Using an Arabidopsis MAPK (mitogen-activated protein kinase) (MPK6) as a model, Sörensson et al. describe in this issue of the Biochemical Journal how arrayed combinatorial peptide scanning offers an efficient route to discovery of new potential kinase substrates.

scholarly journals Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression

1999 ◽  
Vol 19 (1) ◽  
pp. 229-240 ◽  
Author(s):  
Angela M. Cacace ◽  
Neil R. Michaud ◽  
Marc Therrien ◽  
Karen Mathes ◽  
Terry Copeland ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Ras Signaling ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Mitogen Activated Protein

ABSTRACT Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. InXenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

scholarly journals Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).

1991 ◽  
Vol 10 (4) ◽  
pp. 885-892 ◽  
Author(s):  
D. M. Payne ◽  
A. J. Rossomando ◽  
P. Martino ◽  
A. K. Erickson ◽  
J. H. Her ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Regulatory Phosphorylation ◽  
Mitogen Activated Protein

scholarly journals Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages

1997 ◽  
Vol 325 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Jonny WIJKANDER ◽  
Karin GEWERT ◽  
Ulf SVENSSON ◽  
Elisabeth HOLST ◽  
Roger SUNDLER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phorbol Ester ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Kinase C ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity. We have now attempted to map the major phosphorylation sites on the enzyme in such cells. Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increased 32P labelling. Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698–749), containing six serine residues. This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase). Dexamethasone treatment (1–100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria. It remains to be determined whether all phosphorylation sites are equally affected under those conditions.

Phosphorylation of the precursor sequence of rat B-type natriuretic peptide by p34cdc2 and MAP kinase

1994 ◽  
Vol 72 (5-6) ◽  
pp. 227-232 ◽  
Author(s):  
Vasek A. Mezl ◽  
Mark H. Watson ◽  
T. Geoffrey Flynn ◽  
Alan S. Mak
Keyword(s):  
Protein Kinase ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Leader Sequence ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Sea Star ◽  
Mitogen Activated Protein ◽  
Atrial Natriuretic

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), two distinct members of the natriuretic peptide family, share many features in common. However, differences in expression indicate that the processing mechanisms must be different. The leader sequence of rat BNP contains three potential phosphorylation sites for proline-directed kinases that are not present in the leader sequence of ANP. This study has examined how these sites are used by two somewhat different proline-directed kinases. A peptide containing these sites was phosphorylated in vitro by HeLa p34cdc2 kinase and by sea star p44mpk kinase at rates that were comparable to the rates with peptide substrates that are used to assay these enzymes. Sequence analysis of the phosphopeptide shows that both kinases phosphorylate only the two potential phosphorylation sites surrounding the cleavage site of the BNP precursor. The enzymatic potential for such a phosphorylation of BNP in cardiac tissue is demonstrated by immunoblots and kinase assays, showing that in fetal and in adult rat heart both the atria and the ventricles contain a mitogen-activated protein kinase homologue that can phosphorylate this preproBNP sequence.Key words: atrial natriuretic peptide, cdc2 kinase, mitogen-activated protein kinase, phosphorylation.

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