Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Protein Kinase C (PKC) is a family of serine/threonine kinases whose function is influenced by phosphorylation. In particular, three conserved phosphorylation sites known as the activation-loop, the turn-motif and the hydrophobic-motif play important roles in controlling the catalytic activity, stability and intracellular localisation of the enzyme. Prevailing models of PKC phosphorylation suggest that phosphorylation of these sites occurs shortly following synthesis and that these modifications are required for the processing of newly-transcribed PKC to the mature (but still inactive) form; phosphorylation is therefore a priming event that enables catalytic activation in response to lipid second messengers. However, many studies have also demonstrated inducible phosphorylation of PKC isoforms at these sites following stimulation, highlighting that our understanding of PKC phosphorylation and its impact on enzymatic function is incomplete. Furthermore, inducible phosphorylation at these sites is often interpreted as catalytic activation, which could be misleading for some isoforms. Recent studies that include systems-wide phosphoproteomic profiling of cells has revealed a host of additional (and in many cases non-conserved) phosphorylation sites on PKC family members that influence their function. Many of these may in fact be more suitable than previously described sites as surrogate markers of catalytic activation. Here we discuss the role of phosphorylation in controlling PKC function and outline our current understanding of the mechanisms that regulate these phosphorylation sites.     

Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.     

Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm.

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.     

The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation.

BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.     

Phenylarsine oxide and H2O2 plus vanadate induce reverse translocation of phorbol-ester-activated PKCbetaII

The intracellular localization of protein kinase C (PKC) is important for the regulation of its biological activity. Recently, it was reported that, whereas phorbol esters such as PMA induce prolonged translocation of PKC to the plasma membrane, with physiological stimuli, the translocation of PKC is transient and followed by rapid return to the cytoplasm. In addition, this membrane dissociation of PKC was shown to require both the kinase activity of PKC and the phosphorylation of its carboxyl terminus autophosphorylation sites. However, the detailed molecular mechanism of PKC reverse translocation remains obscure. We demonstrated that in porcine polymorphonuclear leucocytes (PMNs), phenylarsine oxide (PAO), a putative protein tyrosine phosphatase (PTPase) inhibitor, induced reverse translocation of PMA-stimulated PKCbetaII. Hydrogen peroxide (H(2)O(2)) in combination with vanadate, both of which are PTPase inhibitors, also induced reverse translocation of PKCbetaII. H(2)O(2) or vanadate alone had little effect on PMA-induced PKCbetaII translocation. Furthermore, genistein and ethanol, which are inhibitors of tyrosine kinase and phospholipase D, respectively, prevented the PKCbetaII reverse translocation induced by the PTPase inhibitors. These results indicate, for the first time, that the tyrosine phosphorylation/phospholipase D pathway may be involved in the process of membrane dissociation of PKC.     

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.     

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.     

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.     

Requirement of protein kinase C micro activation and calpain-mediated proteolysis for arachidonic acid-stimulated adhesion of MDA-MB-435 human mammary carcinoma cells to collagen type IV

Arachidonic acid (AA) stimulation of adhesion of human metastatic breast carcinoma cells to collagen type IV depends on the protein kinase C (PKC) pathway(s) and is associated with the translocation of PKC mu from the cytoplasm to the membrane. In the present study, we have further explored the role of PKC mu in AA-stimulated adhesion. PKC mu activation site serines 738/742 and autophosphorylation site serine 910 are rapidly phosphorylated, and in vitro PKC mu kinase activity is enhanced in response to AA treatment. Inhibition of PKC mu activation blocks AA-stimulated adhesion. A phosphorylated, truncated species of PKC mu was detected in AA-treated cells. This 77-kDa protein contains the kinase domain but lacks a significant portion of the regulatory domains. Inhibition of calpain protease activity blocks generation of the truncated protein, promotes accumulation of the activated, full-length protein in the membrane, and blocks the AA-mediated increase in adhesion. p38 MAPK activity is also required for AA-stimulated adhesion. Activation of PKC mu and p38 are independent events. However, inhibition of p38 activity reduces calpain-mediated proteolysis of PKC mu and in vivo calpain activity, suggesting a role for p38 in regulation of calpain activity and a point for cross-talk between the PKC and MAPK pathways. These results support the hypothesis that AA stimulates activation of PKC mu, which is cleaved by calpain at the cell membrane. The resulting truncated kinase, as well as the full-length kinase, may be required for increased cell adhesion to collagen type IV. Additionally, these studies present the first evidence for calpain cleavage of a non-structural protein leading to the promotion of tumor cell adhesion.     

Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the alpha- and gamma-subunits close to the inner face of the plasma membrane

The alpha-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the alpha-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents. Two novel phosphorylation sites are possible candidates for this PKC phosphorylation: Thr-938 in the M8/M9 loop located very close to the PKA site, and Ser-774, in the proximal part of the M5/M6 hairpin. Both sites are highly conserved in all known alpha-subunits, indicating a physiological role. A similar pattern of detergent-mediated phosphorylation by PKC was found in pig kidney Na,K-ATPase alpha-subunit. Interestingly, the kidney-specific gamma-subunit was phosphorylated by PKC in the presence of detergent. The close proximity of the novel PKC sites to the membrane suggests that targeting proteins to tether PKC into the membrane phase is important in controlling the in vivo phosphorylation of this novel class of membrane-adjacent PKC sites. It is suggested that in purified preparations where functional targeting may be impaired detergents are needed to expose the sites.     

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.     

Catalytic domain crystal structure of protein kinase C-theta (PKCtheta)

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

Background: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.Results: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC βII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC α and βII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.Conclusions: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Parvovirus interference with intracellular signalling: mechanism of PKCeta activation in MVM-infected A9 fibroblasts

Autonomous parvoviruses are strongly dependent on the phosphorylation of the major non-structural protein NS1 by members of the protein kinase C (PKC) family. Besides being accompanied with changes in the overall phosphorylation pattern of NS1 and acquiring new modifications at consensus PKC sites, ongoing minute virus of mice (MVM) infections lead to the appearance of new phosphorylated cellular protein species. This prompted us to investigate whether MVM actively interferes with phosphoinositol-dependent kinase (PDK)/PKC signalling. The activity, subcellular localization and phosphorylation status of the protein kinases PDK1, PKCη and PKCλ were measured in A9 cells in the presence or absence of MVM infection. Parvovirus infection was found to result in activation of both PDK1 and PKCη, as evidenced by changes in their subcellular distribution and overall (auto)phosphorylation. We show evidence that activation of PKCη by PDK1 is driven by atypical PKCλ. By modifying the hydrophobic motif of PKCη, PKCλ appeared to control docking and consecutive phosphorylation of PKCη's activation-loop by PDK1, a process that was inhibited in vivo in the presence of a dominant-negative PKCλ mutant.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.     

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.