The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization.
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Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization. (+info)
Casein kinase I-dependent phosphorylation within a PEST sequence and ubiquitination at nearby lysines signal endocytosis of yeast uracil permease.
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Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. The modification of uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating endocytosis of this protein. This modification in turn facilitates its ubiquitination and internalization. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have previously shown that uracil permease is phosphorylated at several serine residues within a well characterized N-terminal PEST sequence. In this report, we provide evidence that lysine residues 38 and 41, adjacent to the PEST sequence, are the target sites for ubiquitination of the permease. Conservative substitutions at both Lys(38) and Lys(41) give variant permeases that are phosphorylated but fail to internalize. The PEST sequence contains potential phosphorylation sites conforming to the consensus sequences for casein kinase 1. Casein kinase 1 (CK1) protein kinases, encoded by the redundant YCKI and YCK2 genes, are located at the plasma membrane. Either alone supports growth, but loss of function of both is lethal. Here, we show that in CK1-deficient cells, the permease is poorly phosphorylated and poorly ubiquitinated. Moreover, CK1 overproduction rescued the defective endocytosis of a mutant permease in which the serine phosphoacceptors were replaced by threonine (a less effective phosphoacceptor), which suggests that Yck activity may play a direct role in phosphorylating the permease. Permease internalization was not greatly affected in CK1-deficient cells, despite the low level of ubiquitination of the protein. This may be due to CK1 having a second counteracting role in endocytosis as shown by the higher turnover of variant permeases with unphosphorylatable versions of the PEST sequence. (+info)
Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes.
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In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. We propose that the spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth. (+info)
Plasma membrane localization of the Yck2p yeast casein kinase 1 isoform requires the C-terminal extension and secretory pathway function.
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The S. cerevisiae Yck2 protein is a plasma membrane-associated member of the casein kinase 1 protein kinase family that, with its homolog Yck1p, is required for bud morphogenesis, cytokinesis, endocytosis and other cellular processes. Membrane localization of Yckp is critical for its function, since soluble mutants do not provide sufficient biological activity to sustain normal growth. Yck2p has neither a predicted signal sequence nor obvious transmembrane domain to achieve its plasma membrane localization, but has a C-terminal -Cys-Cys sequence that is likely to be palmitoylated. We demonstrate here that Yck2p is targeted through association with vesicular intermediates of the classical secretory pathway. Yck2p lacking C-terminal Cys residues fails to associate with any membrane, whereas substitution of these residues with a farnesyl transferase signal sequence allows sec-dependent plasma membrane targeting and biological function, suggesting that modification is required for interaction with early secretory membranes but that targeting does not require a particular modification. Deletion analysis within the 185 residue C-terminus indicates that the final 28 residues are critical for membrane association, and additional sequences just upstream are required for proper plasma membrane targeting. (+info)
Negative regulation of calcineurin signaling by Hrr25p, a yeast homolog of casein kinase I.
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Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, we screened 119 yeast kinases for their ability to phosphorylate Crz1p in vitro and identified the casein kinase I homolog Hrr25p. Here we show that Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling. (+info)
The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal.
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Our previous work found the two yeast plasma membrane-localized casein kinases Yck1p and Yck2p to be palmitoylated on C-terminal Cys-Cys sequences by the palmitoyl transferase Akr1p. The present work examines a third casein kinase, Yck3p, which ends with the C-terminal sequence Cys-Cys-Cys-Cys-Phe-Cys-Cys-Cys. Yck3p is palmitoylated and localized to the vacuolar membrane. While the C-terminal cysteines are required for this palmitoylation, Akr1p is not. Palmitoylation requires the C-terminal Yck3p residues 463-524, whereas information for vacuolar sorting maps to the 409-462 interval. Vacuolar sorting is disrupted in cis through deletion of the 409-462 sequences and in trans through mutation of the AP-3 adaptin complex; both cis- and trans-mutations result in Yck3p missorting to the plasma membrane. This missorted Yck3p restores 37 degrees C viability to yck1Delta yck2-ts cells. yck1Delta yck2-ts suppressor mutations isolated within the YCK3 gene identify the Yck3p vacuolar sorting signal-the tetrapeptide YDSI, a perfect fit to the YXXPhi adaptin-binding consensus. Although YXXPhi signals have a well-appreciated role in the adaptin-mediated sorting of mammalian cells, this is the first signal of this class to be identified in yeast. (+info)
D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a.
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The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer-Xaa-Xaa-Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1-mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF-1 and insulin. D4476 is much more potent and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1. (+info)
Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I.
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The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved. (+info)