Direct Interaction between Protein Kinase Cθ (PKCθ) and 14-3-3τ in T Cells: 14-3-3 Overexpression Results in Inhibition of PKCθ Translocation and Function†

Abstract

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.

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

“…Transfection of Jurkat T-lymphoblastic Cells with pEF-PKC-His 6 and Purification on Ni 2ϩ Affinity Beads-Jurkat T ag cells are stably transfected with the SV40 large T antigen, which is used to promote expression of genes cloned in the pEF vector (16). pEF-PKC-contains the complete coding region of PKC-and an in-frame His 6 tag at the C terminus.…”

“…Recombinant His 6 -PKC-Phosphorylates Moesin-To verify that PKC-phosphorylates moesin, we transfected eukaryotic cells with a plasmid bearing PKC-with a His 6 tag at its C terminus (16,29). Jurkat (T-lymphoblastic leukemia) cells were transfected with the His 6 -PKC-plasmid and with the neo vector alone.…”

QUICK identification and SPR validation of signal transducers and activators of transcription 3 (Stat3) interacting proteins

“…The 14-3-3 proteins have raised to a position of integrators of diverse signaling cues that impact cell fate and cancer development [31]. Through regulated interactions with crucial signaling mediators, such as PKC [32][33], MAPK [34][35][36], or AKT [37-39], 14-3-3 controls diverse cellular responses ranging from signal transduction, cell cycle, metabolism, and apoptosis [31]. Among seven 14-3-3 proteins, 14-3-3ζ, also termed as YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), has been shown to play a central role regulating multiple pathways responsible for cancer initiation and progression [40].…”

Expression of 14-3-3 ζ and interaction with protein kinase C in the rat retina in early diabetes

“…Many investigators have reported that 14-3-3s act as potential regulators of PKC, which is controlled by the interactions between them [10][11][12][13]. However, the effects of 14-3-3 on PKC have been controversial, showing conflicting results of inhibition or activation [5,9,13], and the biological significance of this event remains unclear.…”

“…Moreover, this isotype is present in significant amounts in the retina, as well as in the brain, and is necessary for light adaptation processes or differentiation in photoreceptors [7,8]. It is well established that 14-3-3 proteins function as a protein kinase C (PKC) regulator [9][10][11][12][13]. In vitro, 14-3-3 ζ has been reported to be an endogenous PKC inhibitor [5,14], but the role of 14-3-3 ζ in the diabetic retina remains unclear.…”