Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.     

Phosphorylation at Ser65 modulates ubiquitin conformational dynamics

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Phosphorylation of spinophilin modulates its interaction with actin filaments

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.     

Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin

Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2.     

Phosphorylation of NBR1 by GSK3 modulates protein aggregation

The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.     

Phosphorylation of NBR1 by GSK3 modulates protein aggregation

The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.     

Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents

Several proteins belonging to the ATP-binding cassettesuperfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1).We measured whole cell swelling-activatedCl currents(I_Cl,swell) inparental cells and cells expressing wild-type MDR1 or aphosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbolester reduced the rate of increase inI_Cl,swell only incells that express MDR1. PKC stimulation had no effect on steady-stateI_Cl,swell.Stimulation of protein kinase A (PKA) with 8-bromoadenosine3',5'-cyclic monophosphate reduced steady-state I_Cl,swell only inMDR1-expressing cells. PKA stimulation had no effect on the rate ofI_Cl,swellactivation. The effects of stimulation of PKA and PKC onI_Cl,swell wereadditive (i.e., decrease in the rate of activation and reduction insteady-stateI_Cl,swell). The effects of PKA and PKC stimulation were absent in cells expressing thephosphorylation-defective mutant. In summary, it is likely thatphosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl channels by independentmechanisms and that Ser-661, Ser-667, and Ser-671 are involved in theresponses ofI_Cl,swell tostimulation of PKA and PKC. These results support the notion that MDR1phosphorylation affectsI_Cl,swell.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.