The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.

It is clear that much remains to be discovered regarding the roles of protein phosphatases in mitogenic signaling pathways. The ability of okadaic acid to activate MAPK/ERKs demonstrates that alteration in serine/threonine dephosphorylation can have significant effects on common steps in growth stimulation induced by different types of mitogens. As in the case of cell cycle control, protein serine/threonine phosphatase plays a central role in the reentry of quiescent cells into the cycle. Because the only known targets of okadaic acid are the catalytic subunits PP1 and PP2A, these enzymes are crucial components of two basic functions carried out by cells: growth and division. Important and obligatory roles for PP2B, PP2C, and newly discovered serine/threonine phosphatases are also likely. However, the limited tissue distribution, unique regulatory properties, and limited substrate specificities of these forms suggest more specialized functions in restricted cell types. The available information on the specific functions of different forms of protein serine/threonine phosphatases, let alone their individual isoforms and different multimeric holoenzymes, is still severely limited. Years of biochemical characterization and cDNA cloning have left us with far more forms than functions. This has led to the gratifying situation, at least for the biochemists, in which genetics and cell biology identify protein phosphatases for which a wealth of biochemical information is already available. The appreciation of the importance of these enzymes in the coming years can only increase as the functions for individual forms are discovered.