A Strategy for the Rapid Identification of Phosphorylation Sites in the Phosphoproteome

MacDonald, Justin A.; Mackey, Aaron J.; Pearson, William R.; Haystead, Timothy A.J.

doi:10.1074/mcp.m200002-mcp200

Cited by 46 publications

(36 citation statements)

References 19 publications

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“…The radiolabeled xPlkk1 was isolated by immunoprecipitation and cleaved with cyanogen bromide. Simultaneous Edman degradation of the labeled peptides was correlated with cycles of 32 P release, and an algorithm analysis was used to assign cycles of 32 P release to specific sites (33). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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A Feedback Loop in the Polo-like Kinase Activation Pathway

Erikson

Haystead

Qian

et al. 2004

Journal of Biological Chemistry

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The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSerhydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G 2 /M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G 2 /M transition.Studies in many laboratories have shown that entry into mitosis is controlled by the activation of cyclin B/Cdc2 following dephosphorylation of Tyr-15 in Cdc2 by the dual specificity phosphatase Cdc25C. Cdc25C itself is activated at the G 2 /M transition by phosphorylation on serine and threonine residues (1-3). Although this activating phosphorylation can be performed by cyclin B/Cdc2 itself (forming a positive feedback loop), evidence in Xenopus eggs and extracts and in mammalian cells suggests that initial activation of Cdc25C depends on phosphorylation by a polo-like kinase (4 -9). The Xenopus pololike kinase Plx1 binds Cdc25C through a noncatalytic domain, the polo box, which is thought to recognize a phosphorylated sequence in Cdc25C (10 -12). Recent evidence (13) suggests that initial phosphorylation of Cdc25C by p38␥ may facilitate binding and activation of Cdc25C by Plx1. However, prior phosphorylation is not obligatory, because Plx1 can efficiently phosphorylate bacterially expressed Cdc25C (see Fig. 2C), which presumably is not phosphorylated. Other studies on the interaction of Plx1 and Cdc25C have shown that overexpression of activated Plx1 accelerates phosphorylation of Cdc25C (14, 15), and depletion of Plx1 from oocyte extracts blocks Cdc25C activation and the G 2 /M transition (6,8). In addition to Cdc25C activation, polo-like kinase is also required for bipolar spindle formation, activation of the anaphase-promoting complex/cyclosome, and cytokinesis (14 -21). Because Plx1 controls both the activation and deactivation of cyclin B/Cdc2, it is a major player in the control of mitosis. This has focus...

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Section: Resultsmentioning

confidence: 99%

“…The results were analyzed by the cleavage of a radiolabeled protein program (fasta.bioch.virginia. edu/crp/) (33).…”

Section: Fig 2 Activation Of Xplkk1 By Plx1mentioning

confidence: 99%

A Feedback Loop in the Polo-like Kinase Activation Pathway

Erikson

Haystead

Qian

et al. 2004

Journal of Biological Chemistry

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“…In the past, phosphorylation analysis was mostly done by radiolabeling with 32/33 P (Bendt et al,. 2003;MacDonald et al,. 2002) combined with amino acid analysis.…”

Section: Phosphoproteome Of Atp Synthasementioning

confidence: 99%

F0F1 ATP Synthase: A Fascinating Challenge for Proteomics

Dabbeni-Sala¹,

Kumar²,

Lippe³

2012

Proteomics - Human Diseases and Protein Functions

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“…Recently, our laboratory has extended the usefulness of phosphorylation site characterization by Edman chemistry through the development of the cleaved radioactive peptide (CRP) program (MacDonald et al, 2002). In CRP analysis, one requires only that the sequence of the protein be known.…”

Section: Phosphorylation Site Identification By Edman Degradationmentioning

confidence: 99%

A Functional Proteomics Approach to Signal Transduction

Graves

Haystead²

2003

Recent Progress in Hormone Research

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The purpose of this review is to highlight how proteomics techniques can be used to answer specific questions related to signal transduction in a wide variety of systems. In our laboratory, we utilize proteomic technologies to elucidate signal transduction pathways involved in smooth muscle contraction and relaxation, cell growth and tumorigenesis, and the pathogenesis of malaria. We see the real application of this technology as a tool to enhance the power of existing approaches such as classical yeast and mouse genetics, tissue culture, protein expression systems, and site-directed mutagenesis. Our basic approach is to examine only those proteins that differ by some variable from the control sample. In this way, the number of proteins to be processed by electrophoresis, Edman degradation, or mass spectrometry is greatly reduced. In addition, since only those proteins that change in response to a given biological treatment are analyzed, the experimental outcome provides information about specific signaling pathways. Examples of typical experiments in our laboratory are measurement of changes in protein phosphorylation in response to treatment of cells with growth factors or specific drugs, characterization of proteins associated with a bait protein in a "pull-down" experiment, or measurement of changes in protein expression. Frequently, in these experiments, it is necessary to define complex protein mixtures. To achieve this goal, we utilize a variety of techniques to isolate specific types of proteins or "subproteomes" for further analysis. In this review, we discuss strategies used in our laboratory for studying signaling pathways, including subproteome isolation, proteome mining, and analysis of the phosphoproteome.

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A Strategy for the Rapid Identification of Phosphorylation Sites in the Phosphoproteome

Cited by 46 publications

References 19 publications

A Feedback Loop in the Polo-like Kinase Activation Pathway

A Feedback Loop in the Polo-like Kinase Activation Pathway

F0F1 ATP Synthase: A Fascinating Challenge for Proteomics

A Functional Proteomics Approach to Signal Transduction

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