Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle

Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

In vitro enhancement of p38 mitogen-activated protein kinase activity by phosphorylated glia maturation factor

We previously demonstrated that glia maturation factor (GMF), a 17-kDa brain protein, can be phosphorylated in test tube by several protein kinases, and that endogenous GMF is rapidly phosphorylated upon stimulation of astrocytes by phorbol 12-myristate 13-acetate. We further observed that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor (IC50 = 3 nM) of the ERK1/ERK2 (p44/p42) subfamily of mitogen-activated protein (MAP) kinase. We now report that, by contrast, PKA-phosphorylated GMF strongly enhances the activity of a related but distinct subfamily of MAP kinase, the p38 MAP kinase, showing an increase of 60-fold over baseline and an EC50 of 7 nM. Non-phosphorylated GMF or GMF phosphorylated by other kinases exhibits only minimal effect. The intracellular interaction of PKA, GMF, and p38 is supported by the phosphorylation of GMF upon cellular stimulation by forskolin (blocked by PKA inhibitor) and by the co-immunoprecipitation of p38 with GMF from cell lysates. Withdrawal of nerve growth factor from PC12 leads to increased GMF phosphorylation with a time course similar to that reported for p38 activation. The results correlate well with a previous report that ERK and p38 carry out opposing functions and implicate GMF as a regulator of major cellular events.     

Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation

The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.     

Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro

The objectives of this study were to evaluate milk choline as an indicator of choline absorption and to use milk choline to evaluate the efficacy of a rumen-protected choline supplement. In a preliminary 4-wk experiment, two Holstein cows in early lactation were abomasally infused with either 0 or 60 g/d of choline chloride in 2 L of water, which was used as a carrier. Choline infusion increased milk choline secretion from 1.95 to 3.95 g/d during the 2-wk choline infusion period. In Experiment 2, four Holstein cows in early lactation were abomasally infused with 0, 25, 50, and 75 g/d of choline chloride in 2 L of water using a 4 x 4 Latin square design with 1-wk experimental periods. Milk choline secretion was 2.56, 3.62, 3.72, and 3.82 g/d for the respective choline treatments. In Experiment 3, 10 Holstein cows in midlactation were fed either 0 or 50 g/d of choline using an experimental rumen-protected choline supplement during a 2-wk experiment. Milk choline secretion was increased from 2.12 to 2.99 g/d with the supplemental choline. Results of these experiments demonstrated that milk choline is responsive to postruminal choline supply and can be used as a qualitative indicator of choline absorption.     

Phorbol ester-induced G1 arrest in BALB/MK-2 mouse keratinocytes is mediated by delta and eta isoforms of protein kinase C

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK-2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA-induced inhibition of DNA synthesis was augmented by overexpression of the eta and delta isoforms, but rescued by the dominant-negative and antisense eta isoforms. In contrast, the alpha and zeta isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle-dependent expression of the eta isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the eta and delta isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.     

Differential activation of protein kinase C delta and epsilon gene expression by gonadotropin-releasing hormone in alphaT3-1 cells. Autoregulation by protein kinase C

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.     

Resting distribution and stimulated translocation of protein kinase C isoforms alpha, epsilon and zeta in response to bradykinin and TNF in human endothelial cells

Protein kinase C (PKC) has been linked to functional and morphological changes in endothelial cells involved in increased microvessel permeability. Bradykinin and TNF are potent inflammatory mediators which translocate PKC from the cytosol to the membrane of various cell types, including endothelial cells. The PKC isoforms alpha, epsilon and zeta have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause increased microvascular permeability via a PKC-dependent endothelial cell signalling pathway. HUVEC were incubated at 37 degrees C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 microM) or TNF (100 U/ml). PMA incubation served as a positive control (100 nM, 15 min). Cytosolic and membrane-bound extracts were obtained by incubation in digitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Western blot and membrane fractions calculated. These experiments revealed that: HUVEC clearly displayed a non-uniform basal membrane fraction distribution of PKC isoforms, with zeta (35.4%) greater than epsilon (30.6%) and both much greater than alpha (8.6%); Bradykinin caused significant translocation of PKC alpha with 15 min and 3 h of treatment but not 1 min; TNF caused dramatic translocation of PKC alpha at 1 min treatment which subsided at 15 min and 3 h but remained significantly elevated; and PMA caused dramatic translocation of alpha and epsilon but not zeta. Treatments of bradykinin and TNF that translocated PKC also showed cytoskeletal rearrangement of rhodamine-phalloidin stained actin, causing it to become more prevalent near cell membranes and concentrated at focal points between cells. These results suggest that PKC alpha may contribute to long term low grade increases in microvessel permeability in response to bradykinin, and that PKC alpha could be involved in both transient and sustained microvessel permeability changes induced by TNF. Also, cytoskeletal actin organization appears to be a downstream pathway in the activation process, possibly leading to alteration in endothelial cell shape and contact points.     

Transforming growth factor-beta enhances the ultraviolet-mediated stress response in p53-/- keratinocytes

This review focuses on the contributions of modern mass spectrometry to neuropeptide research. An introduction to newer mass spectrometric techniques is provided. Also, the use of mass spectrometry in combination with high-resolution separation techniques for neuropeptide identification in biological samples is illustrated. The amino acid sequence information that is important for the identification and analysis of known, novel, or chemically modified neuropeptides may be obtained using mass spectrometric techniques. Because mass spectrometry techniques can be used to reflect the dynamic properties associated with neuropeptide processing in biological systems, they may be used in the future to monitor peptide profiles within organisms in response to environmental challenges such as disease and stress.     

14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.