Functional implications of post-translational modifications of phospholipases D1 and D2

Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(DeltaN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(DeltaN168) when coexpressed in COS 7 cells and that palmitoylation of Cys(240) and Cys(241) was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys(223) and Cys(224) in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(DeltaN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.     

Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.     

Association of the N- and C-terminal domains of phospholipase D. Contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)(4)D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998) J. Biol. Chem. 273, 34679-34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCalpha and small G proteins in vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

Protein kinase C inhibits type VI adenylyl cyclase by phosphorylating the regulatory N domain and two catalytic C1 and C2 domains

We previously showed that phosphorylation of Ser(10) of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitro phosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser(568) and Ser(674) of the C1 domain and Thr(931) of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser(10), Ser(568), Ser(674), and Thr(931)) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser(10), Ser(568), and Ser(674)) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr(931) was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr(931) to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI.     

Hierarchy of membrane-targeting signals of phospholipase D1 involving lipid modification of a pleckstrin homology domain

The amino terminus of phospholipase D1 (PLD1) contains three potential membrane-interacting determinants: a phox homology (PX) domain, a pleckstrin homology (PH) domain and two adjacent cysteines at positions 240 and 241 within the PH domain that are fatty acylated in vivo. To understand how these determinants contribute to membrane localization, we have mutagenized critical residues of the PLD1 PH domain in the wild type or palmitate-free background in the intact protein, in a fragment that deletes the first 210 amino acids including the PX domain, and in the isolated PH domain. Mutants were expressed in COS-7 cells and examined for membrane residence, intracellular localization, palmitoylation, and catalytic activity. Our results are as follows. 1) Mutagenesis of critical residues of the PH domain results in redistribution of PLD1 from membranes to cytosol, independently of fatty acylation sites. Importantly, PH domain mutants in the wild type background showed greatly reduced fatty acylation, despite the presence of all relevant cysteines. 2) The isolated PH domain did not co-localize with PLD1 and was not palmitoylated. 3) The PX deletion mutant showed similar distribution and palmitoylation to the intact protein. Interestingly, PH domain mutants in this background showed significant palmitoylation and incomplete cytosolic redistribution. 4) PH domain mutants in the wild type or palmitate-free background maintained catalytic activity. We propose that membrane targeting of PLD1 involves a hierarchy of signals with a functional PH domain allowing fatty acylation leading to strong membrane binding. The PX domain may modulate function of the PH domain.     

Phosphorylation of human inhibitor-1 at Ser67 and/or Thr75 attenuates stimulatory effects of protein kinase A signaling in cardiac myocytes

The depressed function of failing hearts has been partially attributed to increased protein phosphatase-1 through its impaired regulation by inhibitor-1. Phosphorylation of inhibitor-1 at Thr35 by PKA results in potent inhibition of protein phosphatase-1 activity, while phosphorylation at Ser67 or Thr75 by PKC attenuates the inhibitory activity. To examine the functional role of dual-site (Ser67, Thr75) phosphorylation of inhibitor-1 by PKC, the constitutively phosphorylated Ser67 (S67D) and/or Thr75 (T75D) human inhibitor-1 forms were expressed in adult cardiomyocytes. Expression of either single or double phosphorylated inhibitor-1 was associated with similar decreases in cardiac contractility, indicating that maximal inhibition can be elicited by each of these sites alone and that their inhibitory effects are not additive. Notably, activation of the cAMP pathway could only partially reverse the depressed contractile parameters. Accordingly, protein phosphatase-1 activity remained elevated, phosphorylation of phospholamban at Ser16 was decreased, and the EC(50) values of the sarcoplasmic reticulum calcium transport system were higher compared with controls. Thus phosphorylation of Ser67 and/or Thr75 in inhibitor-1 may mitigate the stimulatory effects of the cAMP pathway, resulting in compromised cardiac function.     

Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.     

Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.     

Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.     

Dual regulation of platelet protein kinase B

Protein kinase B (PKB) is a serine/threonine kinase that is activated by growth hormones and implicated in prevention of apoptosis, glycogen metabolism, and glucose uptake. A key enzyme in PKB activation is phosphatidylinositide 3-kinase (PI-3K), which triggers the dual phosphorylation of PKB by phosphatidylinositol-dependent kinases (PDKs). Here we report that the major PKB subtype in platelets is PKBalpha, which is activated by phosphorylation of Thr(308) and Ser(473) and has a constitutively phosphorylated Thr(450) that does not contribute to PKB activation. alpha-Thrombin and thrombopoietin activate PKBalpha via PI-3K and trigger the concurrent phosphorylation of Thr(308) (via PDK1) and Ser(473) (via a not yet identified PDK2). In addition, alpha-thrombin activates a PI-3K-independent pathway involving phospholipase Cbeta and calcium-dependent protein kinase C subtypes (PKCalpha/beta). This route is specific for phosphorylation of Ser(473) and can be initiated by direct PKC activation with phorbol ester or purified active PKC catalytic fragment in platelet lysate. Different degrees of Ser(473) and Thr(308) phosphorylation correlate with different degrees of enzyme activity. These data reveal a PI-3K-independent PKB activation in which PKCalpha/beta regulates the phosphorylation of Ser(473) in PKBalpha. The independent control of the two phosphorylation sites may contribute to fine regulation of PKBalpha activity.     

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.     

mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.     

Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase

A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.     

Two C-terminal amino acids, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of opioid receptor-like 1 receptors

Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CDelta21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CDelta34), which differs with ORL1(CDelta21) by lacking C-terminal Ser(334), Ser(335) and Ser(343) residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser(334) or Ser(335) with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser(334) and Ser(335) also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of ORL1.     

Protein kinase C regulates the activity and stability of serotonin N-acetyltransferase

Effects of protein kinase C on protein stability and activity of rat AANAT were investigated in vitro and in vivo. When COS-7 cells transfected with AANAT cDNA were treated with phorbol 12-myristate 13-acetate (PMA), both the activity and protein level of AANAT were increased. These effects of PMA were blocked by GF109203X, a specific inhibitor of PKC. Moreover, PMA increased the phosphorylation of AANAT and induced the formation of AANAT/14-3-3zeta complex. PMA did not affect the basal level of cAMP and did not involve the potentiation of the cAMP production by forskolin, indicating that PKC-dependent activation of adenylyl cyclase was excluded in transfected COS-7 cells. To identify which amino acids were phosphorylated by PKC, several conserved Thr and Ser residues in AANAT were targeted for site-directed mutagenesis. Mutations of Thr29 and Ser203 prevented the increase of enzymatic activity and protein level mediated by PMA. To explore the nature of AANAT phosphorylation, purified rat AANAT was subjected to in vitro PKC kinase assay. PKC directly phosphorylated the rat recombinant AANAT. The phosphopeptides identified by mass spectrometric analysis, and western blotting indicated that Thr29 was one of target sites for PKC. To confirm the effects of the physiological activation of PKC, rat pineal glands were treated with alpha(1)-adrenergic specific agonist phenylephrine. Phenylephrine caused the phosphorylation of endogenous AANAT whereas GF109203X or prazosin, an alpha(1)-adrenergic-specific antagonist, markedly inhibited it. These results suggest that AANAT was phosphorylated at Thr29 by PKC activation through the alpha(1)-adrenergic receptor in rat pineal glands, and that its phosphorylation might contribute to the stability and the activity of AANAT.     

Primary structure of the casein macropeptide of caprine kappa casein]

The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and "thermolysin" fragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated. Asp2, Asn5, Thr11, Ser6, SerP2, Glu7, Gln2, Pro6, Ala9 Val5, Met1, Ile6, Lys3, His1, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons. The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier [28, 27]. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.     

PKCdelta-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCdelta on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCdelta-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCdelta catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.     

Protein kinase C-mediated down-regulation of cyclin D1 involves activation of the translational repressor 4E-BP1 via a phosphoinositide 3-kinase/Akt-independent, protein phosphatase 2A-dependent mechanism in intestinal epithelial cells

We reported previously that protein kinase Calpha (PKCalpha), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCalpha-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr(45) and Ser(64) on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCalpha-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an approximately 2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H(2)O(2) and ceramide, two naturally occurring PKCalpha agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCalpha-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCalpha-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.