Casein kinase 1-dependent phosphorylation of familial advanced sleep phase syndrome-associated residues controls PERIOD 2 stability.

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.

Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43-kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300-400-kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes but retained FUS/TLS binding activity. The functional significance of TDP-43-FUS/TLS complexes was established by showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression of histone deacetylase (HDAC) 6 mRNA. TDP-43 and FUS/TLS associated with HDAC6 mRNA in intact cells and in vitro, and competition experiments suggested that the proteins occupy overlapping binding sites. The combined findings demonstrate that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may reflect their participation in common biochemical processes.

Non-specific in vivo inhibition of CK1 by the pyridinyl imidazole p38 inhibitors SB 203580 and SB 202190.

Small-molecule inhibitors of protein kinases have contributed immensely to our understanding of biological signaling pathways and have been exploited therapeutically for the treatment of cancers and other disease states. The pyridinyl imidazole compounds SB 203580 and SB 202190 were identified as ATP competitive antagonists of the p38 stress-activated protein kinases and have been widely used to elucidate p38-dependent cellular processes. Here, we identify SB 203580 and SB 202190 as potent inhibitors of stress-induced CREB phosphorylation on Serine 111 (Ser-111) in intact cells. Unexpectedly, we found that the inhibitory activity of SB 203580 and SB 202190 on CREB phosphorylation was independent of p38, but instead correlated with inhibition of casein kinase 1 (CK1) in vitro. The inhibition of CK1-mediated CREB phosphorylation by concentrations of pyridinyl imidazoles commonly employed to suppress p38, suggests that in some cases conclusions of p38-dependence derived solely from the use of these inhibitors may be invalid.

Potentiation of amyotrophic lateral sclerosis (ALS)-associated TDP-43 aggregation by the proteasome-targeting factor, ubiquilin 1.

TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates.