Regulation of angiotensin II-induced G protein signaling by phosducin-like protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP(3)) formation, whereas further increases caused an abrupt decrease in IP(3) production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gbetagamma. Results of pertussis toxin sensitivity, GTPgammaS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cbeta activation by dual mechanisms: (i) steric blockage of Gbetagamma activation of PLCbeta and (ii) interference with Gbetagamma-dependent cycling of G(q)alpha by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.     

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.     

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.     

Regulation of protein kinase C-related protein kinase 2 (PRK2) by an intermolecular PRK2-PRK2 interaction mediated by Its N-terminal domain

Protein kinase C-related protein kinases (PRKs) are effectors of the Rho family of small GTPases and play a role in the development of diseases such as prostate cancer and hepatitis C. Here we examined the mechanism underlying the regulation of PRK2 by its N-terminal region. We show that the N-terminal region of PRK2 prevents the interaction with its upstream kinase, the 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates the activation loop of PRK2. We confirm that the N-terminal region directly inhibits the kinase activity of PRK2. However, in contrast to previous models, our data indicate that this inhibition is mediated in trans through an intermolecular PRK2-PRK2 interaction. Our results also suggest that amino acids 487-501, located in the linker region between the N-terminal domains and the catalytic domain, contribute to the PRK2-PRK2 dimer formation. This dimerization is further supported by other N-terminal domains. Additionally, we provide evidence that the region C-terminal to the catalytic domain intramolecularly activates PRK2. Finally, we discovered that the catalytic domain mediates a cross-talk between the inhibitory N-terminal region and the activating C-terminal region. The results presented here describe a novel mechanism of regulation among AGC kinases and offer new insights into potential approaches to pharmacologically regulate PRK2.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

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.     

Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit complex (Gbetagamma) that has been recently shown to catalyze the formation of the Gbetagamma dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbetagamma dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbetagamma-assembly process. The results reported here delineate a mechanism for Gbetagamma assembly in which a stable ternary complex is formed between PhLP, the nascent Gbeta subunit, and CCT that does not include Ggamma. PhLP phosphorylation permits the release of a PhLP x Gbeta intermediate from CCT, allowing Ggamma to associate with Gbeta in this intermediate complex. Subsequent interaction of Gbetagamma with membranes releases PhLP for another round of assembly.     

Regulation of arrestin-3 phosphorylation by casein kinase II

Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.     

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.     

Regulation of CHK2 by DNA-dependent protein kinase

Chk2 is a critical mediator of diverse cellular responses to DNA damage. Activation of Chk2 by DNA damage requires phosphorylation at sites including Thr68. In earlier work, we found that an activity present in rabbit reticulocyte lysates phosphorylates and activates Chk2. We now find that hypophosphorylated Chk2 can be phosphorylated at Thr68 by various subcellular fractions of HEK293 cells. This activity is sensitive to the phosphatidylinositol 3'-kinase-like kinase inhibitor wortmannin, but not to caffeine. DNA enhances the Chk2 phosphorylation by cellular fractions in vitro. The wortmannin-sensitive Chk2 kinase activity is present in fractions from ATM-deficient cells. In contrast, Chk2 was not efficiently phosphorylated at Thr68 in vitro by fractions from cells with a defective DNA-dependent protein kinase (DNA-PK) catalytic subunit. Chk2 is phosphorylated by purified DNA-PK in vitro. Endogenous Chk2 coimmunoprecipitates Ku70 and Ku80. In a series of matched cell lines having and lacking functional DNA-PK, Chk2 activation by exposure of cells to ionizing radiation, or to camptothecin was consistently diminished in the absence of DNA-PK. Down-regulation of DNA-PK(cs) by either siRNA or a chemical inhibitor attenuated radiation-induced Chk2 phosphorylation. Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage.     

Regulation of the Drosophila melanogaster protein, enhancer of rudimentary, by casein kinase II

The Drosophila melanogaster gene enhancer of rudimentary, e(r), encodes a conserved protein, ER. Most ER homologs share two casein kinase II (CKII) target sites. In D. melanogaster, these sites are T18 and S24. A third CKII site, T63, has been seen only in drosophilids. The conservation of these CKII sites, particularly T18 and S24, suggests a role for these residues in the function of the protein. To test this hypothesis, these positions were mutated either to alanine as a nonphosphorylated mimic or to glutamic acid as a phosphorylated mimic. The mutations were tested individually or in double or triple combinations for their ability to rescue either a wing truncation characteristic of the genotype e(r)(p1) r(hd1-12) or the synthetic lethal interaction between e(r)(p2) and the Notch allele N(nd-p). All of the substitutions as single mutations rescued both mutant phenotypes, arguing that individually the phosphorylation of the three residues does not affect ER activity. The double mutants T18A-S24A and T18E-S24E and the triple mutants T18A-S24A-T63A and T18E-S24E-T63E failed to rescue. Together the data support the following model for the regulation of ER by CKII. ER that is unphosphorylated at both T18A and S24 is inactive. CKII activates ER by phosphorylating either T18 or S24. Further phosphorylation to produce the doubly phosphorylated protein inactivates ER.     

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.     

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.     

Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.     

Phosphorylation of casein by cyclic AMP-dependent protein kinase.

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α_s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.