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Identification and characterization of neuronal mitogen-activated protein kinase substrates using a specific phosphomotif antibody.

Edbauer D, Cheng D, Batterton MN, Wang CF, Duong DM, Yaffe MB, Peng J, Sheng M - Mol. Cell Proteomics (2008)

Bottom Line: We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner.Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons.Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

View Article: PubMed Central - PubMed

Affiliation: The Picower Institute for Learning and Memory, Howard Hughes Medical Institute, RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in delta-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

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In vitro phosphorylation of candidate substrates by mitogen-activated protein kinases. Selected candidate proteins (GFP-δ-catenin, GFP-PKP4, FLAG-4.1N, FLAG-AbLIM1, FLAG-MAGI-2, FLAG-SAPAP4, FLAG-PIP5K1C, and FLAG-SynGAP1) were expressed in HEK293 cells, immunoprecipitated with epitope tag antibodies, and incubated with γ-[P32]ATP and different recombinant active MAPKs as indicated. Phosphorylation was visualized by autoradiography. FLAG-tagged transcription factor Elk-1 is used as positive control (see “Results”).
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f5: In vitro phosphorylation of candidate substrates by mitogen-activated protein kinases. Selected candidate proteins (GFP-δ-catenin, GFP-PKP4, FLAG-4.1N, FLAG-AbLIM1, FLAG-MAGI-2, FLAG-SAPAP4, FLAG-PIP5K1C, and FLAG-SynGAP1) were expressed in HEK293 cells, immunoprecipitated with epitope tag antibodies, and incubated with γ-[P32]ATP and different recombinant active MAPKs as indicated. Phosphorylation was visualized by autoradiography. FLAG-tagged transcription factor Elk-1 is used as positive control (see “Results”).

Mentions: As a further test we asked whether the proteins identified from the 5557 phosphoantibody immunoprecipitation could serve as substrates for MAPKs in vitro. A set of candidate proteins were epitope-tagged, expressed in HEK293 cells, and then immunoprecipitated as substrates for in vitro kinase reactions with recombinant ERK2 or JNK1 and radioactive [γ-32P]ATP (Fig. 5). As a positive control we used the transcription factor Elk-1, which is a known substrate of ERK and JNK (34). ERK2 and JNK1 both readily phosphorylated Elk-1, δ-catenin, PKP4, and to a lesser extent 4.1N, AbLIM1, Adducin 1, MAGI-2, and SAPAP4. Thus our phosphoantibody immunoprecipitation yielded many MAPK substrates at least as defined by in vitro phosphorylation. We found no evidence for phosphorylation of SynGAP1 (synaptic GTPase-activating protein 1) and PIP5K1C (phosphatidylinositol-4-phosphate 5-kinase, type I, γ) by ERK2 or JNK1, although both of them were identified from PSD by 5557 immunoprecipitation. However, it is possible that these proteins are substrates of p38 or other ERK/JNK isoforms not tested here.


Identification and characterization of neuronal mitogen-activated protein kinase substrates using a specific phosphomotif antibody.

Edbauer D, Cheng D, Batterton MN, Wang CF, Duong DM, Yaffe MB, Peng J, Sheng M - Mol. Cell Proteomics (2008)

In vitro phosphorylation of candidate substrates by mitogen-activated protein kinases. Selected candidate proteins (GFP-δ-catenin, GFP-PKP4, FLAG-4.1N, FLAG-AbLIM1, FLAG-MAGI-2, FLAG-SAPAP4, FLAG-PIP5K1C, and FLAG-SynGAP1) were expressed in HEK293 cells, immunoprecipitated with epitope tag antibodies, and incubated with γ-[P32]ATP and different recombinant active MAPKs as indicated. Phosphorylation was visualized by autoradiography. FLAG-tagged transcription factor Elk-1 is used as positive control (see “Results”).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667352&req=5

f5: In vitro phosphorylation of candidate substrates by mitogen-activated protein kinases. Selected candidate proteins (GFP-δ-catenin, GFP-PKP4, FLAG-4.1N, FLAG-AbLIM1, FLAG-MAGI-2, FLAG-SAPAP4, FLAG-PIP5K1C, and FLAG-SynGAP1) were expressed in HEK293 cells, immunoprecipitated with epitope tag antibodies, and incubated with γ-[P32]ATP and different recombinant active MAPKs as indicated. Phosphorylation was visualized by autoradiography. FLAG-tagged transcription factor Elk-1 is used as positive control (see “Results”).
Mentions: As a further test we asked whether the proteins identified from the 5557 phosphoantibody immunoprecipitation could serve as substrates for MAPKs in vitro. A set of candidate proteins were epitope-tagged, expressed in HEK293 cells, and then immunoprecipitated as substrates for in vitro kinase reactions with recombinant ERK2 or JNK1 and radioactive [γ-32P]ATP (Fig. 5). As a positive control we used the transcription factor Elk-1, which is a known substrate of ERK and JNK (34). ERK2 and JNK1 both readily phosphorylated Elk-1, δ-catenin, PKP4, and to a lesser extent 4.1N, AbLIM1, Adducin 1, MAGI-2, and SAPAP4. Thus our phosphoantibody immunoprecipitation yielded many MAPK substrates at least as defined by in vitro phosphorylation. We found no evidence for phosphorylation of SynGAP1 (synaptic GTPase-activating protein 1) and PIP5K1C (phosphatidylinositol-4-phosphate 5-kinase, type I, γ) by ERK2 or JNK1, although both of them were identified from PSD by 5557 immunoprecipitation. However, it is possible that these proteins are substrates of p38 or other ERK/JNK isoforms not tested here.

Bottom Line: We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner.Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons.Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

View Article: PubMed Central - PubMed

Affiliation: The Picower Institute for Learning and Memory, Howard Hughes Medical Institute, RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in delta-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

Show MeSH
Related in: MedlinePlus