Physiological role for casein kinase 1 in glutamatergic synaptic transmission.
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Casein kinase 1 (CK1) is a highly conserved serine/threonine kinase, present in virtually all cell types, in which it phosphorylates a wide variety of substrates. So far, no role has been found for this ubiquitous protein kinase in the physiology of nerve cells. In the present study, we show that CK1 regulates fast synaptic transmission mediated by glutamate, the major excitatory neurotransmitter in the brain. Through the use of CK1 inhibitors, we present evidence that activation of CK1 decreases NMDA receptor activity in the striatum via a mechanism that involves activation by this kinase of protein phosphatase 1 and/or 2A and resultant increased dephosphorylation of NMDA receptors. Indeed, inhibition of CK1 increases NMDA-mediated EPSCs in medium spiny striatal neurons. This effect is associated with an increased phosphorylation of the NR1 and NR2B subunits of the NMDA receptor and is occluded by the phosphatase inhibitor okadaic acid. The mGluR1, but not mGluR5, subclass of metabotropic glutamate receptors uses CK1 to inhibit NMDA-mediated synaptic currents. These results provide the first evidence for a role of CK1 in the regulation of synaptic transmission in the brain. (+info)
BCR kinase phosphorylates 14-3-3 Tau on residue 233.
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The breakpoint cluster region protein, BCR, has protein kinase activity that can auto- and trans-phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14-3-3 is of major interest. 14-3-3tau and zeta isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14-3-3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14-3-3. In this study we have further characterized the BCR-14-3-3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14-3-3 in vivo and phosphorylates 14-3-3tau on Ser233 and to a lesser extent 14-3-3zeta on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14-3-3zeta. (+info)
Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.
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Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells. (+info)
IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508.
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Interleukin (IL)-2 is the predominant cytokine that is produced by naive Th cells in a primary response. It is required for proliferation and differentiation of Th precursor cells into effector cells. Initial high-level IL-2 production is followed by its decline, and the concomitant induction of cytokines that are typical of the differentiated state. Although the factors that are responsible for the early induction of IL-2 are well defined, the mechanisms that are responsible for its down-regulation in later stages of Th development have not been studied as much. Previous work from our laboratory revealed a repressor function for the T-box transcription factor, T-bet, in IL-2 gene transcription. Here, we report that T-bet(S508) is required for the optimal repression of IL-2 production in developing Th1 cells. Phosphorylation of T-bet(S508) by casein kinase I and glycogen synthase kinase-3 kinases accompanies T-bet's interaction with the RelA nuclear factor-kappaB transcription factor. Heterodimerization of T-bet and RelA interferes with the binding of RelA to the IL-2 promoter, and hence, transcriptional activation of the IL-2 gene by RelA. (+info)
The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8.
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Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3/Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuole-associated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate/palmitate (as in Vac8) but also one that is targeted by a myristate/basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole. (+info)
Glc7-Reg1 phosphatase signals to Yck1,2 casein kinase 1 to regulate transport activity and glucose-induced inactivation of Saccharomyces maltose permease.
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The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1delta yck2-2(ts) (yck(ts)) strain with conditional Yck activity. Moreover, we provide evidence that Glc7-Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yck(ts) strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yck(ts) strain and maltose permease proteolysis is blocked. We show that a reg1delta mutant exhibits a phenotype remarkably similar to that conferred by yck(ts). The reg1delta phenotype is not enhanced in the yck(ts) reg1delta double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1delta mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1delta strain. (+info)
The identification of a functional interaction between PKC and topoisomerase II.
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Topoisomerase II plays an essential role in the segregation of chromosomes during cell division. It is also a major component of the nuclear matrix. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between the catalytic domain of the yeast protein kinase 1 enzyme (Pkc1) and the N-terminal domain of the S. cerevisiae topoisomerase II. The S. cerevisiae Pkc1 is the homologue of the mammalian calcium dependent PKC. (+info)
Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing.
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Hedgehog-regulated processing of the transcription factor cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by casein kinase 1 and glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. Here we show that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Our gain- and loss-of-function analyses in cultured cells further reveal that betaTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and we demonstrate that betaTrCP can bind phosphorylated Gli3 both in vitro and in vivo. We also find that the Gli3 protein is polyubiquitinated in the cell and that its processing depends on proteasome activity. Our findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and betaTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome. (+info)