Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines.

The molecular mechanisms regulating the ubiquitin proteasome system (UPS) at synapses are poorly understood. We report that CaMKIIalpha-an abundant postsynaptic protein kinase-mediates the activity-dependent recruitment of proteasomes to dendritic spines in hippocampal neurons. CaMKIIalpha is biochemically associated with proteasomes in the brain. CaMKIIalpha translocation to synapses is required for activity-induced proteasome accumulation in spines, and is sufficient to redistribute proteasomes to postsynaptic sites. CaMKIIalpha autophosphorylation enhances its binding to proteasomes and promotes proteasome recruitment to spines. In addition to this structural role, CaMKIIalpha stimulates proteasome activity by phosphorylating proteasome subunit Rpt6 on Serine 120. However, CaMKIIalpha translocation, but not its kinase activity, is required for activity-dependent degradation of polyubiquitinated proteins in spines. Our findings reveal a scaffolding role of postsynaptic CaMKIIalpha in activity-dependent proteasome redistribution, which is commensurate with the great abundance of CaMKIIalpha in synapses.

Proline-rich tyrosine kinase 2 regulates hippocampal long-term depression.

Proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase beta or protein tyrosine kinase 2b, is a calcium-dependent signaling protein involved in cell migration. Phosphorylation of residue Y402 is associated with activation of PYK2 and leads to the recruitment of downstream signaling molecules. PYK2 was previously implicated in long-term potentiation (LTP); however, the role of PYK2 in long-term depression (LTD) is unknown. Here, we report that PYK2 is activated by NMDA receptor stimulation (chemical LTD) in cultured neurons. Small hairpin RNA-mediated knockdown of PYK2 blocks LTD, but not LTP, in hippocampal slice cultures. We find that the Y402 residue and, to a lesser extent, PYK2 kinase activity contribute to PYK2's role in LTD. Knockdown experiments indicate that PYK2 is required to suppress NMDA-induced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of PYK2 depresses NMDA-induced ERK phosphorylation and inhibits LTP, but not LTD. Our data indicate that PYK2 is critical for the induction of LTD, possibly in part by antagonizing ERK signaling in hippocampal neurons.

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.

Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures.

Bioengineering approaches, such as co-cultures of multiple cell types, that aim to mimic the physiological microenvironment may be beneficial for optimizing cell function and for engineering tissues in vitro. This study describes a novel method for preparing a spheroid microarray on microfabricated hydrogels, alone or in co-cultures. Photocrosslinkable chitosan was synthesized and utilized for fabricating hydrogel microstructures through a micromolding process. The chitosan surface was initially cell repellent but became increasingly cell adhesive over time. By using this unique property of chitosan hydrogels, it was possible to generate patterned co-cultures of spheroids and support cells. In this scheme, cells were initially microarrayed within low shear stress regions of microwells. Human hepatoblastoma cells, Hep G2, seeded in these wells formed spheroids with controlled sizes and shapes and stably secreted albumin during the culture period. The change of cell adhesive properties in the chitosan surface facilitated the adhesion and growth of a second cell type, NIH-3T3 fibroblast, and therefore enabled co-cultures of hepatocyte spheroids and fibroblast monolayers. This co-culture system could be a useful platform for studying heterotypic cell-cell interactions, for drug screening, and for developing implantable bioartificial organs.

Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132.

MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

Diversity and complexity in DNA recognition by transcription factors.

Sequence preferences of DNA binding proteins are a primary mechanism by which cells interpret the genome. Despite the central importance of these proteins in physiology, development, and evolution, comprehensive DNA binding specificities have been determined experimentally for only a few proteins. Here, we used microarrays containing all 10-base pair sequences to examine the binding specificities of 104 distinct mouse DNA binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in the evolution of transcriptional regulatory networks.