Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

Protein kinase C signaling is desensitized through a combination of dephosphorylation and proteolysis in intact cells. The process of dephosphorylation is analyzed here, as well as its relationship to degradation. It is established for protein kinase C␣ that dephosphorylation occurs in a membrane compartment following activation and temporally preceding significant degradation. The phosphatase responsible for the dephosphorylation appears to be a heterotrimeric type 2A phosphatase, which is shown to be in part constitutively membrane associated. Consistent with a role for this activity, okadaic acid is shown to inhibit the phorbol ester-induced dephosphorylation of protein kinase C that occurs in intact cells. Furthermore, phorbol esterinduced down-regulation of protein kinase C␣ is shown not to be dependent on the rate of dephosphorylation, indicating that these desensitizing pathways may operate in parallel.

The generation of antisera specific for the priming phosphorylation sites on protein kinase Calpha (PKCalpha) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCalpha is able to stimulate the dephosphorylation of wild-type PKCalpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCalpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCalpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCbeta1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.

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Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.

Bombesin induces the down-regulation of protein kinase C-delta (PKC-delta) and PKC-epsilon in Swiss 3T3 cells. Simultaneous addition of transforming growth factor beta 1 (TGF beta 1) selectively blocks PKC-delta down-regulation at mid-S-phase, whereas PKC-epsilon levels continue to decline. Northern blot analysis shows that PKC-epsilon levels could be controlled in part at the level of transcription; PKC-delta mRNA levels remained constant at these later times. Bombesin induces a sustained elevation of some species of diacylglycerol (DAG), consistent with the observed loss of PKC-delta and PKC-epsilon. Interestingly, the combination of bombesin and TGF-beta 1 produces an even greater DAG response. Flow cytometric analysis demonstrates that bombesin induces only 15% of the cells to enter the cell cycle, in contrast to the combination of TGF beta 1 plus bombesin which induces 75-80% of the cells to progress through the cycle. The protection of PKC-delta from down-regulation under conditions of sustained DAG elevation correlates with the mitogenic response and implies that the down-regulation process itself is regulated. Consistent with this, it is demonstrated that bombesin plus TGF beta 1 protects PKC-delta from phorbol ester-induced down-regulation.

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