Identification and characterization of neuronal mitogen-activated protein kinase substrates using a specific phosphomotif antibody.
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.
Edbauer
Dieter
D
The Picower Institute for Learning and Memory, Howard Hughes Medical Institute, RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Cheng
Dongmei
D
Batterton
Matthew N
MN
Wang
Chi-Fong
CF
Duong
Duc M
DM
Yaffe
Michael B
MB
Peng
Junmin
J
Sheng
Morgan
M
D020816:Amino Acid Motifs; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism*; D051177:Catenins; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism; D047468:Immunoprecipitation; D007553:Isotope Labeling; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D013379:Substrate Specificity / Q000187:drug effects; D000095002:Delta Catenin
D045163:Antibodies, Phospho-Specific; D051177:Catenins; D015815:Cell Adhesion Molecules; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C033652:postsynaptic density proteins; D010768:Phosphoserine; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000095002:Delta Catenin