Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o−/− mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and β isoforms in the brain. Although CaMKIIα functions have been elucidated, the unique catalytic functions of CaMKIIβ have remained unknown. Using knockdown analyses in primary neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 localizes CaMKIIβ at the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC as a novel centrosomal substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a novel isoform-specific function for CaMKIIβ that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.

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