Protein Kinase C-dependent Phosphorylation Research Articles

Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser187Potentiates Vesicle Recruitment

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

Receptor for activated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABA(A) receptors with the activation of G-protein-coupled receptors.

GABA(A) receptors are the principal sites of fast synaptic inhibition in the brain. These receptors are hetero-pentamers that can be assembled from a number of subunit classes: alpha(1-6), beta(1-3), gamma(1-3), delta(1), epsilon, theta;, and pi, but the majority of receptor subtypes is believed, however, to be composed of alpha, beta, and gamma2 subunits. A major mechanism for modulating GABA(A) receptor function occurs via the phosphorylation of residues within the intracellular domains of receptor subunits by a range of serine/threonine and tyrosine kinases. However, how protein kinases are targeted to these receptors to facilitate functional modulation remains unknown. Here we demonstrate that the receptor for activated C kinase (RACK-1) and protein kinase C (PKC) bind to distinct sites on GABA(A) receptor beta subunits. Although RACK-1 is not essential for PKC binding to GABA(A) receptor beta subunits, it enhances the phosphorylation of serine 409, a residue critical for the phospho-dependent modulation of GABA(A) receptor function in the beta1 subunit by anchored PKC. Furthermore, RACK-1 also enhances GABA(A) receptor functional modulation in neurons by a PKC-dependent signaling pathway with the activation of muscarinic acetylcholine receptors (mAChRs). This PKC-dependent modulation of neuronal GABA(A) receptors was mirrored by an increase in the phosphorylation of GABA(A) receptor beta subunits with the activation of mAChRs. Our results suggest a central role for RACK-1 in potentiating PKC-dependent phosphorylation and functional modulation of GABA(A) receptors. Therefore, RACK-1 will enhance functional cross talk between GABA(A) receptors and G-protein-coupled receptors and therefore may have profound effects on neuronal excitability.

Receptor for Activated C Kinase-1 Facilitates Protein Kinase C-Dependent Phosphorylation and Functional Modulation of GABAAReceptors with the Activation of G-Protein-Coupled Receptors

GABA_A receptors are the principal sites of fast synaptic inhibition in the brain. These receptors are hetero-pentamers that can be assembled from a number of subunit classes: α(1–6), β(1–3), γ(1–3), δ(1), ε, θ, and π, but the majority of receptor subtypes is believed, however, to be composed of α, β, and γ2 subunits. A major mechanism for modulating GABA_A receptor function occurs via the phosphorylation of residues within the intracellular domains of receptor subunits by a range of serine/threonine and tyrosine kinases. However, how protein kinases are targeted to these receptors to facilitate functional modulation remains unknown. Here we demonstrate that the receptor for activated C kinase (RACK-1) and protein kinase C (PKC) bind to distinct sites on GABA_A receptor β subunits. Although RACK-1 is not essential for PKC binding to GABA_A receptor β subunits, it enhances the phosphorylation of serine 409, a residue critical for the phospho-dependent modulation of GABA_Areceptor function in the β1 subunit by anchored PKC. Furthermore, RACK-1 also enhances GABA_A receptor functional modulation in neurons by a PKC-dependent signaling pathway with the activation of muscarinic acetylcholine receptors (mAChRs). This PKC-dependent modulation of neuronal GABA_A receptors was mirrored by an increase in the phosphorylation of GABA_A receptor β subunits with the activation of mAChRs. Our results suggest a central role for RACK-1 in potentiating PKC-dependent phosphorylation and functional modulation of GABA_A receptors. Therefore, RACK-1 will enhance functional cross talk between GABA_A receptors and G-protein-coupled receptors and therefore may have profound effects on neuronal excitability.

Protein Kinase C and Guanosine Triphosphate Combine to Potentiate Calcium-dependent Membrane Fusion Driven by Annexin 7

Exocytotic secretion is promoted by the concerted action of calcium, guanine nucleotide, and protein kinase C. We now show that the calcium-dependent membrane fusion activity of annexin 7 in vitro is further potentiated by the combined addition of guanine nucleotide and protein kinase C. The observed increment involves the simultaneous activation of annexin 7 by these two effectors. Guanosine triphosphate (GTP) and its non-hydrolyzable analogues optimally enhance the phosphorylation of annexin 7 by protein kinase C in vitro. Reciprocally, phosphorylation by protein kinase C significantly potentiates the binding and hydrolysis of GTP by annexin 7. Only protein kinase C-dependent phosphorylation has a significant positive effect on annexin 7 GTPase, although other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and pp60(c-)(src), have been shown to label the protein with high efficiency. In vivo, the ratio of bound GDP/GTP and phosphorylation of annexin 7 change in direct proportion to the extent of catecholamine release from chromaffin cells in response to stimulation by carbachol, or to inhibition by various protein kinase C inhibitors. These results thus lead us to hypothesize that annexin 7 may serve as a common site of action for calcium, guanine nucleotide, and protein kinase C in the exocytotic membrane fusion process in chromaffin cells.

The Golgi Localization of Phosphatidylinositol Transfer Protein β Requires the Protein Kinase C-dependent Phosphorylation of Serine 262 and Is Essential for Maintaining Plasma Membrane Sphingomyelin Levels

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function.

Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

Effect of oleoresin capsicum (OC) and ortho-chlorobenzylidene malononitrile (CS) on ciliary beat frequency

Tear gases are largely used to control civil unrest. Their incapaciting effects involve the eyes, skin, and respiratory tract. We aimed to evaluate the effects of ortho-chlorobenzylidene malononitrile (CS) and oleoresin capsicum (OC) on ciliary beat frequency (CBF) of mouse tracheal rings. Addition of 0.05% OC or 0.01% CS induced a progressive decrease in CBF, from 11.5±0.5 to 4±0.1 Hz ( P<0.05) and from 12.5±0.5 to 2.5±0.1 Hz ( P<0.05), respectively, 30 min after exposure to the tear gas. Addition of exogenous ATP inhibited the effect of OC, suggesting that ATP could be used to counteract these adverse effects on CBF. However, ATP was inefficient against CS. Methylene blue and H7 inhibited the effects of OC, whereas indomethacin had no effect. None of these drugs affected the inhibitory action of CS. These results suggest that the inhibitory effect of OC is mediated through the guanylate cyclase-dependant pathway or protein kinase C-dependent phosphorylation. Another mechanism is probably involved in CS-induced inhibitory effect. Histological analysis of the trachea revealed an increase in mucus secretion after exposure to OC, and cytoplasmic vacuoles in epithelial cells after exposure to CS.

CAMP-dependent Protein Kinase Induces cAMP-response Element-binding Protein Phosphorylation via an Intracellular Calcium Release/ERK-dependent Pathway in Striatal Neurons

Activation of the cAMP-dependent protein kinase A (PKA) pathway may induce cAMP-response element-binding protein (CREB) phosphorylation either directly or via cross-talk mechanisms with other signal transduction pathways. In this study, we have investigated in striatal primary cultures the mechanism by which activation of the cAMP/PKA-dependent pathway leads to CREB phosphorylation via the extracellular signal-regulated kinase (ERK)-dependent pathway. We have found that PKA-induced CREB phosphorylation and CREB-dependent transcription are mediated by calcium (Ca(2+)) release from intracellular stores and are blocked by inhibitors of the protein kinase C and ERK pathways. This mechanism appears to be mediated by the small G-protein Rap1, whose activation appears to be primed by PKA-induced Ca(2+) release but not further induced by direct or indirect PKA- or protein kinase C-dependent phosphorylation. These results suggest that, in striatal neurons, intracellular Ca(2+) release, Rap1, and ERK pathway play a crucial role in the PKA-induced CREB phosphorylation and CREB-dependent transcription.

Better signaling through chemistry

Thromboxane A2 (TxA2), a potent platelet aggregator and vasoconstrictor, is produced by cyclooxygenase-mediated cleavage of arachidonic acid. Its cellular effects are mediated through a Gq protein-coupled receptor (TP), leading to protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. However, the mechanism of the latter effect has been poorly characterized. Relying on previous studies, which suggest that epidermal growth factor receptor (EGFR) is the link between Gq/Gi protein-coupled receptors and MAPK activation, Gao et al.1xThe Thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor. Gao, Y. et al. J. Pharm. Exp. Ther. 2001; 296: 426–433PubMedSee all References1 embark on a ‘pharmacological and molecular’ dissection of the TxA2–MAPK signaling pathway. Specifically, this study illuminates a network of interactions between a G protein-coupled receptor, phosphoinositide, and a growth factor receptor pathway. More importantly, however, the methods illustrate the principles of chemical genetics, and portend the promise of this new field.Armed with an arsenal of activators and inhibitors of TP, Gi, PKC, EGFR, and Src tyrosine kinase, the authors investigate the effects of these modulators on MAPK phosphorylation. Starting with the TxA2 agonist IBOP, they show that treatment of a human bladder cancer cell line with certain inhibitors attenuates IBOP-stimulated MAPK phosphorylation. Working backwards toward the physiological stimulus, they demonstrate that IBOP-mediated phosphorylation of EGFR is suppressed by each inhibitor, suggesting that the EGFR is upstream of MAPK but downstream of Gi, PKC, and Src. The role of Src in TP-dependent activation of MAPK was further tested using a dominant negative Src construct. The authors demonstrate that, in response to IBOP, both Src kinase activity and MAPK phosphorylation are stimulated either in the presence or the absence of an inhibitor of EGFR, confirming a place for Src upstream of the EGFR in this network. Using the agonists LPA and PMA, Gao et al. investigate the roles of Gi and PKC, respectively, in MAPK phosphorylation. Calphostin C, a PKC inhibitor, has no effect on LPA stimulation, whereas each other inhibitor reduces MAPK phosphorylation, albeit slightly. Pursuing the role of PKC in TP-Gi signaling, the authors note an inhibitor-mediated decrease in TP-G?i interaction, as measured by immunoprecipitation and Western blot. Gao et al. close by modeling the PKC most proximal to the TxA2 receptor itself and as the primary facilitator of Src activity, EGFR autophosphorylation and MAPK phosphorylation.In classical genetics, the relationships between the elements of a signal transduction network are studied using epistatic analysis, whereby a sequence of biological events is inferred by comparing single and multiple gene deletions with the wild-type state. By analogy, chemical epistatic analysis is the process of establishing such a relationship using small molecules, rather than mutations, to alter protein function. Small molecules, as well as temperature-sensitive mutations, provide the additional advantage of temporal control over protein function. Gao et al. have successfully applied these principles to shed new light on the interaction between a G protein-coupled receptor and the MAPK cascade. In particular, the positioning of Src upstream of EGFR represents an important departure from conventional wisdom. As the number of ligands that modulate protein function increases, we will probably see similar studies that employ chemical epistatic analysis to define network relationships in signal transduction.

Regulation of the Ligand Binding Activity of the Human Very Low Density Lipoprotein Receptor by Protein Kinase C-dependent Phosphorylation

The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.

Determinants of G protein inhibition of presynaptic calcium channels.

The modulation of presynaptic calcium (Ca) channels by heterotrimeric G proteins is a key factor for the regulation of neurotransmission. Over the past 20 yr, a significant understanding of the molecular events underlying this regulation has been acquired. It is now widely accepted that binding of G protein betagamma dimers directly to the cytoplasmic region linking domains I and II of the Ca channel alpha1 subunit results in a stabilization of the closed conformation of the channel, thereby inhibiting current activity. The extent of the inhibition is dependent on the Gbeta subunit isoform, and is antagonized by both strong membrane depolarizations and protein kinase C-dependent phosphorylation of the channel. Finally, the inhibition is critically modulated by regulator of G protein signaling proteins, and by proteins forming the presynaptic vesicle release complex. Thus, the regulation of the activities of presynaptic Ca channels is becoming increasingly complex, a feature that may contribute to the overall fine-tuning of Ca entry into presynaptic nerve termini, and thus, neurotransmission.

Bradykinin-regulated Interactions of the Mitogen-activated Protein Kinase Pathway with the Endothelial Nitric-oxide Synthase

Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC). Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-(32)P]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro (32)P phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 μM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 μM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the β isoform of phosphatidylinositol 3-kinase regulatory subunit.

The affinity of the organic cation transporter rOCT1 is increased by protein kinase C-dependent phosphorylation.

Members of the organic cation transporter (OCT) family are mainly expressed in kidney, liver, intestine, and brain. The regulation of the OCT type 1 from rat (rOCT1) stably transfected in HEK293 cells was examined using a fluorimetric technique, 1-[(3)H]methyl-4-phenylpyridinium uptake studies, and fast-whole-cell patch-clamp recordings. For the fluorescence measurements, the cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was used as substrate. Uptake of ASP(+) via rOCT1 was electrogenic, and its inhibition by other organic cations was consistent with previously reported radioactive tracer flux measurements. The inhibitor quinine was not translocated by the organic cation transporter in contrast to tetraethylammonium. Stimulation of diacyl glycerol-dependent protein kinase C (PKC) by sn-1,2-dioctanoyl glycerol (1 microM) resulted in an increase in initial ASP(+) uptake rate by 216 +/- 28% (n = 29). The effect was completely antagonized by the PKC inhibitor tamoxifen (20 microM, n = 22). Forskolin (1 microM), which activates adenylate cyclase and thereby protein kinase A (PKA), stimulated the initial rate of ASP(+) accumulation by 51 +/- 6% (n = 19). This effect was inhibited by the specific PKA inhibitor KT5720 (1 microM, n = 12). Inhibition of tyrosine kinases by aminogenestein (10 microM) reduced ASP(+) uptake by 63 +/- 7% (n = 7), while genestein or tyrphostin AG1295 (each 10 microM) were without significant effects. Incubation of the cells with sn-1, 2-dioctanoyl glycerol (1 microM) increased the affinities of the transporter to tetraethylammonium, tetrapenthylammonium, and quinine by a factor of 58, 14.5, and 2.4, respectively. Western blot analysis revealed that rOCT1 protein was phosphorylated at a serine residue upon stimulation of PKC. In conclusion, it has been demonstrated that the organic cation transport by rOCT1 is stimulated by PKC, PKA, and endogenous tyrosine kinase activation. The PKC phosphorylates rOCT1 and leads to a conformational change at the substrate binding site.

The Protein Kinase C-dependent Phosphorylation of Serine 166 Is Controlled by the Phospholipid Species Bound to the Phosphatidylinositol Transfer Protein α

The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.

The F-actin cytoskeleton modulates slow secretory components rather than readily releasable vesicle pools in bovine chromaffin cells

Adrenal chromaffin cells were used to test the role of the peripheral cytoskeleton of F-actin in controlling different vesicle pools. Phorbol 12-myristate 13-acetate and calyculin A, two substances affecting phosphorylation–dephosphorylation cycles, produced different degrees of F-actin reorganization, inducing the partial and the almost total disassembly of this structure, respectively, as visualized using rhodamine–phalloidin staining. Consequently, electron microscopy studies revealed the higher efficiency of calyculin-A over phorbol 12-myristate 13-acetate in promoting vesicle access to the plasmalemma boundary. Surprisingly, only the phorbol ester enhanced fast kinetics and the population of rapidly releasable vesicle pools as studied by single-cell amperometry, whereas both agents, as well as the F-actin severing compound, Latrunculin A, promoted an increase in the population of vesicles recruited in response to prolonged or repetitive stimulations. Taken together, our data support the notion that the F-actin peripheral barrier controls primary granule recruitment from reserve vesicle pools, whereas the phorbol ester effect on the rapidly releasable pools might be related to the alteration of late secretory stage through protein kinase C-dependent phosphorylation of an unidentified target.

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking.

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.

MARCKS-related Protein (MRP) Is a Substrate for the Leishmania major Surface Protease Leishmanolysin (gp63)

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Metalloprotease-Disintegrin MDC9: Intracellular Maturation and Catalytic Activity

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-alpha convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-alpha-convertase.