Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.     

Inhibition of exendin-4-induced steatosis by protein kinase A in cultured HepG2 human hepatoma cells

Nonalcoholic fatty liver is characterized by the abnormal accumulation of triglycerides within hepatocytes, resulting in a steatotic liver. Glucagon-like peptide 1 and its analog exendin-4 can ameliorate certain aspects of this syndrome by inducing weight loss and reducing hepatic triglyceride accumulation, but it is unclear whether these effects result from the effects of glucagon-like peptide 1 on the pancreas, or from direct action on the liver. This study investigated the direct action and putative cellular mechanism of exendin-4 on steatotic hepatocytes in culture. Steatosis was induced in cultured HepG2 human hepatoma cells by incubation in media supplemented with 2 mM each of linoleic acid and oleic acid. Steatotic hepatocytes were then pre-incubated in the protein kinase A inhibitor H89 for 30 min, then treated with exendin-4 over a period of 24 h. Cell viability and triglyceride content were characterized by a TUNEL assay and AdipoRed staining, respectively. Our results showed that steatotic cells maintained high levels of intracellular triglycerides (80%) compared to lean controls (25%). Exendin-4 treatment caused a significant reduction in intracellular triglyceride content after 12 h that persisted through 24 h, while protein kinase A inhibitors abolished the effects of exendin-4. The results demonstrate the exendin-4 induces a partial reduction in triglycerides in steatotic hepatocytes within 12 h via the GLP-1 receptor-mediated activation of protein kinase A. Thus, the reduction in hepatocyte triglyceride accumulation is likely driven primarily by downregulation of lipogenesis and upregulation of β-oxidation of free fatty acids.     

Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling

Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.     

Inhibition of casein kinase 1-epsilon induces cancer-cell-selective,PERIOD2-dependent growth arrest

Background  

Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.     

Phosphorylation of protein kinase C by casein kinase-1

Because phosphorylation of protein kinase C (PKC) may provide a mechanism for regulation of this enzyme, we have examined the ability of two other kinases to phosphorylate PKC. Our results show that casein kinase 1 (CK-1), but not casein kinase 2 (CK-2), can phosphorylate PKC in the absence of Ca2+ and phospholipids. The 32P incorporation into PKC in the presence of Ca2+ and phospholipids is also enhanced by CK-1.     

La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells

The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+-affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP-IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+, indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+.     

M-phase-specific cdc2 protein kinase phosphorylates the beta subunit of casein kinase II and increases casein kinase II activity

The M-phase-specific cdc2 (cell division control) protein kinase (a component of the M-phase-promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5-5-fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non-hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The beta subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the beta subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M-phase and therefore be involved in the pleiotropic effects of M-phase-promoting factor.     

Pancreatitis-associated protein 2 modulates inflammatory responses in macrophages

Pancreatitis-associated proteins (PAP) are stress-induced secretory proteins that are implicated in immunoregulation. Previous studies have demonstrated that PAP is up-regulated in acute pancreatitis and that gene knockdown of PAP correlated with worsening severity of pancreatitis, suggesting a protective effect for PAP. In the present study, we investigated the effect of PAP2 in the regulation of macrophage physiology. rPAP2 administration to clonal (NR8383) and primary macrophages were followed by an assessment of cell morphology, inflammatory cytokine expression, and studies of cell-signaling pathways. NR8383 macrophages which were cultured in the presence of PAP2 aggregated and exhibited increased expression of IL-1, IL-6, TNF-alpha, and IL-10; no significant change was observed in IL-12, IL-15, and IL-18 when compared with controls. Chemical inhibition of the NFkappaB pathway abolished cytokine production and PAP facilitated nuclear translocation of NF-kappaB and phosphorylation of IkappaB alpha inhibitory protein suggesting that PAP2 signaling involves this pathway. Cytokine responses were dose dependent. Interestingly, similar findings were observed with primary macrophages derived from lung, peritoneum, and blood but not spleen. Furthermore, PAP2 activity was inhibited by the presence of serum, inhibition which was overcome with increased PAP2. Our results demonstrate a new function for PAP2: it stimulates macrophage activity and likely modulates the inflammatory environment of pancreatitis.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

Phosphorylation of maize RAB-17 protein by casein kinase 2

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.     

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.