Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin

In this study we identified snapin as an interaction partner of the CK1 isoform delta (CK1delta) in the yeast two-hybrid system and localized the interacting domains of both proteins. The interaction of CK1delta with snapin was confirmed by co-immunoprecipitation. Snapin was phosphorylated by CK1delta in vitro. Both proteins localized in close proximity in the perinuclear region, wherein snapin was found to associate with membranes of the Golgi apparatus. The identification of snapin as a new substrate of CK1delta points towards a possible function for CK1delta in modulating snapin specific functions.     

Identification of novel protein kinase CK1 delta (CK1delta) inhibitors through structure-based virtual screening

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer's disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).     

Blocking of the effect of delta1 tetrahydrocannabinol (delta1THC) on spontaneous motor activity in the rat by immunization with a protein conjugate of delta1THC

Rats actively immunized with porcine gamma globulin- hemisuccinate-Δ¹-tetrahydrocannabinol (PγG-HS-Δ¹THC) showed higher spontaneous motor activity after intraperitoneal administration of Δ¹THC at a dose of 10 mg. per kg. than did rats immunized with a control antigen, porcine gamma globulin-hemisuccinate-phenol (PγG-HS-Phenol). The capacity to neutralize the effect of Δ¹THC was found to depend on the degree of immunization; thus, the difference in mean spontaneous motor activity after injection of Δ¹THC was significant in rats which had received five injections of the immunogen over a period of 86 days, and not in those which had received only two injections over a period of 34 days.In view of the observations that Δ¹-tetrahydrocannabinol induces a decrease in spontaneous motor activity in rats, the observed neutralization of the effect of δ¹THC in animals receiving multiple injections of protein conjugates of Δ¹THC may be due to the binding of the drug by anti-THC antibodies (which are expected to be produced on active immunization with these conjugates), thus preventing Δ¹THC from reaching drug-receptor sites.     

Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.     

Repeated delta1-opioid receptor stimulation reduces delta2-opioid receptor responses in the SA node

Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via delta(1)-opioid receptors within the sinoatrial (SA) node. Higher doses activate delta(2)-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the delta(1)-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended delta(1)-opioid receptor stimulation reduces subsequent delta(2)-receptor responses. The delta(2)-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate delta(2) responses before and after infusion of the delta(1)-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the delta(1)-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by delta(1) activity. When TAN-67 was omitted in time control studies, some loss of delta(2) responses was apparent in the absence of the delta(1) treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of delta(1) activity. Additional studies tested TAN-67 alone in the absence of prior deltorphin. When time controls were conducted without the initial deltorphin treatment, a robust vagolytic response was observed. When TAN-67 preceded the delayed deltorphin, the vagolytic response was eroded, indicating an independent effect of TAN-67. BNTX infused afterward was unable to restore the delta(2) response. These data support the conclusion that the loss of the delta(2) response resulted from reduced delta(2) activity mediated by continued delta(1)-receptor stimulation and not the arithmetic consequence of increased competition from that same delta(1) receptor.     

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization

Mammalian casein kinase 1delta (CK1delta) is a homologue of the S. cerevisiae Hrr25p protein kinase. Hrr25p is involved in regulating diverse events including vesicular trafficking, gene expression, DNA repair, and chromosome segregation. In contrast to Hrr25p, little is known about the function, regulation, or subcellular localization of CK1delta. In the present study, we show that CK1delta in mammalian cells is mainly cytoplasmic and enriched within the Golgi and/or ER-Golgi transport vesicles, consistent with a role in vesicular trafficking. Transient expression of green fluorescent protein (GFP)- or FLAG peptide-tagged CK1delta showed localization similar to that of the endogenous CK1delta. GFP-CK1delta was also enriched at the centrosomes in interphase cells. Strikingly, two inactive mutant CK1delta proteins (K38M and T176I) showed almost exclusive nuclear staining, suggesting that protein kinase activity is required for normal localization of CK1delta and prevention of nuclear accumulation. The nuclear export inhibitor leptomycin B promoted nuclear enrichment of CK1delta indicating that nuclear localization of CK1delta occurs physiologically. Both endogenous CK1delta and GFP-CK1delta are enriched on the spindle poles in mitotic cells, consistent with a role in regulating spindle formation. Localization is a property of the protein kinase domain and is independent of the C-terminal noncatalytic domain. These data are consistent with roles for CK1delta in mammalian cells analogous to those of its yeast counterparts.     

Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)

CK1delta, a member of the casein kinase 1 family of serine/threonine specific kinases, has been shown to be involved in the regulation of microtubule dynamics. We have now identified a 176 aa fragment of the light chain LC2 of MAP1A (termed LC2-P16) specifically interacting with CK1delta. Two CK1delta interacting domains of LC2 were identified, located between aa 2629 and 2753 close to aa 2683 and between aa 2712 and 2805 of LC2. The two regions necessary for the interaction of LC2 with CK1delta have been mapped between aa 76-103 and aa 351-375 of CK1delta. Furthermore, LC2 has been identified as a new substrate of CK1delta. We therefore propose a model in which CK1delta could modulate microtubule dynamics by changing the phosphorylation status of the light chain LC2 of MAP1A.     

Identification of nuclear factor delta EF1 and its binding site essential for lens-specific activity of the delta 1-crystallin enhancer.

The lens-specific reglatory element of the delta 1-crystallin enhancer lies within the core segment (Goto et al., (1990) Mol. Cell. Biol. 10, 935-964). The element was allocated within the 55 bp long HN fragment of the core. Block-wise base substitutions were introduced to the 55 bp and their effect on the enhancer activity of the multimers in lens cells was examined. By base sequence alteration of either of the contiguous blocks 5 and 6, with their original sequence of TTGCT and CACCT, respectively, enhancer activity was totally lost. A lens nuclear factor delta EF1 was found which bound specifically to the base sequences defined by the blocks. DNA binding activity very similar to delta EF1 was also found in extracts of tissues other than lens, suggesting that delta EF1 participates in lens-specific regulation through tissue-dependent modification or interaction with other factors.     

The elongation factor-1delta (EF-1delta) originates from gene duplication of an EF-1beta ancestor and fusion with a protein-binding domain.

The molecular evolution of two components of elongation factor-1 (EF-1), EF-1beta and EF-1delta was analysed using the distance matrix, the maximum parsimony and the maximum likelihood methods, after careful alignment of protein and cDNA sequences. The topology of the phylogenetic trees obtained supports monophyly of plant EF-1beta and EF-1beta' sequences, and monophyly of higher eukaryotic animal EF-1beta and EF-1delta sequences. EF-1beta and EF-1delta are homologous in their C-terminal domain. EF-1delta, which emerged before arthropods, originates from a beta-type ancestor gene and fusion with a leucine zipper N-terminal motif. Plant EF-1beta and EF-1beta' correspond to paralogous genes whose ancestor was most likely duplicated before the emergence of monocotyledons and dicotyledons.     

JAGGED1 and delta1 differentially regulate the outcome of experimental autoimmune encephalomyelitis

Notch signaling plays an important role during T cell development in the thymus and in T cell activation but the role of Notch in autoimmunity is not clear. We investigated the role of Jagged1 and Delta1 in experimental autoimmune encephalomyelitis. During experimental autoimmune encephalomyelitis, Delta1 expression is up-regulated on dendritic cells and B cells after priming while Jagged1 is up-regulated only on dendritic cells. Administration of anti-Jagged1 Ab exacerbated clinical disease while that of anti-Delta1 Ab reduced the severity of the clinical disease. In contrast, administration of Jagged1-Fc protected from disease, that of Delta1-Fc exacerbated disease. Treatment with Jagged1-Fc was associated with increased IL-10-producing Ag-specific cells in the CNS, while anti-Jagged1 decreased the frequency of IL-10-producing cells. Treatment with Delta1-Fc increased Th1 cells in the CNS, while anti-Delta-1 decreased the frequency of Th1 cells. Manipulation of Delta1 or Jagged1 had no effect on the frequency of Th17 cells or FoxP3(+) cells. Moreover, Jagged1 may play a role in CNS homeostasis because murine astrocytes specifically express Jagged1 that is up-regulated by TGF-beta, whereas IFN-gamma, TNF-alpha, and IL-17 decrease Jagged1 expression. Our study provides novel data about differential roles of Notch ligands in regulating inflammation in the periphery as well as in the CNS.     

delta1-piperideine-2-carboxylate reductase of Pseudomonas putida.

Pseudomonas putida metabolizes D-lysine to delta 1-piperideine-2-carboxylate and L-pipecolate. The second step of this catabolic pathway is catalyzed by delta 1-piperideine-2-carboxylate reductase. This enzyme was isolated and purified from cells grown on DL-lysine as substrate. The enzyme was very unstable, resulting in low recovery of activity and low purity after a six-step purification procedure. The enzyme had a pH optimum of 8.0 to 8.3. The Km values for delta 1-piperideine-2-carboxylate and NADPH were 0.23 and 0.13 mM, respectively. NADPH at concentrations above 0.15 mM was inhibitory to the enzyme. Delta 1-pyrroline-5-carboxylate, pyroglutamate, and NADH were poor substrates or coenzyme for delta 1-piperideine-2-carboxylate reductase. The enzyme reaction from delta 1-piperideine-2-carboxylate to L-pipecolate was irreversible. EDTA, sodium pyrophosphate, and dithiothreitol at concentrations of 1 mM protected the enzyme during storage. The enzyme was inhibited almost totally by Zn2+, Mn2+, Hg2+ Co2+, and p-chloromercuribenzoate at concentrations of 0.1 mM. The enzyme had a molecular weight of about 200,000. Both D-lysine and L-lysine were good inducers for the enzyme. Neither delta1-piperideine-2-carboxylate nor L-pipecolate was an effective inducer for the enzyme. P. putida cells grew on D-lysine only after a 5- to 8-h lag, which could be abolished by adding a supplement of 0.01% alpha-ketoglutarate or other readily metabolizable compounds. Such a supplement also converted the noncoordinate induction of this enzyme and pipecolate oxidase, both of the D-lysine pathway, to coordinacy. However, this effect was not observed if the enzyme pair was from different pathways of lysine metabolism in this organism (i.e., the D- and L-lysine pathways).