c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro ; definition of the minimal kinase recognition motif (original) (raw)
Related papers
Analysis of the Interaction between c-Jun and c-Jun N-terminal Kinase in Vivo
Journal of Biological Chemistry, 1998
Regulation of c-Jun transcriptional activity is believed to depend on a physical interaction with c-Jun N-terminal kinase (JNK) that facilitates signal-regulated phosphorylation of multiple regulatory phosphoacceptor sites within the activation domain. Here we have investigated the structural requirements and consequences of regulatory phosphorylation for the interaction between c-Jun and JNK in vivo. We show that binding of JNK to c-Jun in vivo does not require JNK catalytic activity or the presence of the potential phosphoacceptor sites within c-Jun and that JNK retains the capacity to bind to a pseudo-phosphorylated mutant of c-Jun where these sites are replaced by phospho-mimetic aspartic acid residues. The c-Jun delta region docking site is essential for interaction with JNK in vivo but is not sufficient, because a c-Jun mutant that retains this region but that lacks the C-terminal DNA-binding domain fails to interact. Experiments using purified recombinant c-Jun and JNK proteins show that the c-Jun DNA-binding domain harbors an auxiliary interaction domain that has the potential to bind to JNK independently. Our results suggest that JNK can be tethered passively to c-Jun in situ through multiple interacting regions and, when activated, can stimulate c-Jun phosphorylation without necessarily dissociating from its substrate. Auxiliary interactions mediated by the DNA-binding domain could play a role in targeting JNK preferentially to c-Jun in specific homo-or heterodimeric complexes.
The EMBO journal, 1994
Protein phosphorylation is commonly used to modulate transcription factor activity. However, all existing genetic evidence for stimulation of transcription factor activity by phosphorylation rests on loss-of-function mutations. To demonstrate conclusively that phosphorylation of a transcription factor potentiates its transactivation potential in vivo, we constructed a c-Jun mutant that is phosphorylated by the cAMP-sensitive protein kinase A (PKA) instead of the UV- and Ras-responsive protein kinase JNK. The transcriptional activity of this mutant is enhanced by PKA, but not by JNK activation. These results provide a positive and conclusive proof that phosphorylation of c-Jun on a critical site (Ser73) located in its activation domain is directly responsible for enhancing its transactivation function.
Genes & Development, 1993
The activity of c-Jun is regulated by phosphorylation. Various stimuli including transforming oncogenes and UV light, induce phosphorylation of serines 63 and 73 in the amino-terminal activation domain of c-Jun and thereby potentiate its trans-activation function. We identified a serine/threonine kinase whose activity is stimulated by the same signals that stimulate the amino-terminal phosphorylation of c-Jun. This novel c-Jun amino-terminal kinase (JNK), whose major form is 46 kD, binds to a specific region within the c-Jun trans-activation domain and phosphorylates serines 63 and 73. Phosphorylation results in dissociation of the c-Jun-JNK complex. Mutations that disrupt the kinase-binding site attenuate the response of c-Jun to Ha-Ras and UV. Therefore the binding of JNK to c-Jun is of regulatory importance and suggests a mechanism through which protein kinase cascades can specifically modulate the activity of distinct nuclear targets.
Genes & Development, 1994
The transcriptional activity of c-Jun is augmented through phosphorylation at two sites by a c-Jun amino-terminal kinase (JNK). All cells express two distinct JNK activities, 46 and 55 kD in size. It is not clear which of them is the more important c-Jun kinase and how they specifically recognize c-Jun. The 46-kD form of JNK was identified as a new member of the MAP kinase group of signal-transducing enzymes, JNK1. Here, we report the molecular cloning of the 55-kD form of JNK, JNK2, which exhibits 83% identity and similar regulation to JNK1. Despite this close similarity, the two JNKs differ greatly in their ability to interact with c-Jun. JNK2 binds c-Jun approximately 25 times more efficiently than JNK1, and as a result has a lower Km toward c-Jun than JNK1. The structural basis for this difference was investigated and traced to a small beta-strand-like region near the catalytic pocket of the enzyme. Modeling suggests that this region is solvent exposed and therefore is likely to serve as a docking site that increases the effective concentration of c-Jun near JNK2. These results explain how two closely related MAP kinases can differ in their ability to recognize specific substrates and thereby elicit different biological responses.
Molecular and cellular biology, 1999
POU domain proteins have been implicated as key regulators during development and lymphocyte activation. The POU domain protein T-cell factor beta1 (TCFbeta1), which binds octamer and octamer-related sequences, is a potent transactivator. In this study, we showed that TCFbeta1 is phosphorylated following activation via the T-cell receptor or by stress-induced signals. Phosphorylation of TCFbeta1 occurred predominantly at serine and threonine residues. Signals which upregulate Jun kinase (JNK)/stress-activated protein kinase activity also lead to association of JNK with TCFbeta1. JNK associates with the activation domain of TCFbeta1 and phosphorylates its DNA binding domain. The phosphorylation of recombinant TCFbeta1 by recombinant JNK enhances the ability of TCFbeta1 to bind to a consensus octamer motif. Consistent with this conclusion, TCFbeta1 upregulates reporter gene transcription in an activation- and JNK-dependent manner. In addition, inhibition of JNK activity by catalytical...
POU domain proteins have been implicated as key regulators during development and lymphocyte activa- tion. The POU domain protein T-cell factor b1 (TCFb1), which binds octamer and octamer-related sequences, is a potent transactivator. In this study, we showed that TCFb1 is phosphorylated following activation via the T-cell receptor or by stress-induced signals. Phosphorylation of TCFb1 occurred predominantly at serine and threonine residues. Signals which upregulate Jun kinase (JNK)/stress-activated protein kinase activity also lead to association of JNK with TCFb1. JNK associates with the activation domain of TCFb1 and phosphor- ylates its DNA binding domain. The phosphorylation of recombinant TCFb1 by recombinant JNK enhances the ability of TCFb1 to bind to a consensus octamer motif. Consistent with this conclusion, TCFb1 upregulates reporter gene transcription in an activation- and JNK-dependent manner. In addition, inhibition of JNK activity by catalytically inactive MEKK (in wh...
c-Jun Regulates Phosphoinositide-dependent Kinase 1 Transcription
Journal of Biological Chemistry
Mutations in N-RAS and B-RAF, which commonly occur in melanomas, result in constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling. Active ERK increases expression and activity of the c-Jun transcription factor, linking ERK and Jun N-terminal kinase (JNK) cascades. Here, we show that c-Jun regulates transcription of phosphoinositide-dependent kinase 1 (PDK1) with a concomitant impact on Akt and protein kinase C (PKC) activity and related substrates. Inhibition of c-Jun reduces PDK1 expression and attenuates Akt and PKC activity, which can be restored by exogenous PDK1. c-Jun regulation of PDK1 in melanoma contributes to growth rate and the ability to form tumors in mice. Correspondingly, increased levels of c-Jun in melanoma cell lines coincide with up-regulation of PDK1 and phosphorylation of PKC and Akt. The identification of c-Jun as a transcriptional regulator of PDK1 expression highlights key mechanism...
Multiple signal transduction pathways mediate c-Jun protein phosphorylation
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1993
A variety of protein kinases, including pp42 and pp54 mitogen-activated protein (MAP) kinases, p34cdc2, and a partially purified protein kinase from 4 beta-phorbol 12-myristate 13 alpha-acetate (PMA)-treated U937 cells have been shown to phosphorylate the NH2-terminal activation domain of c-Jun in vitro. To investigate the role of pp42 MAP kinase in mediating c-Jun phosphorylation in vivo, we have treated U937 monocytic leukemia cells with a variety of pharmacological agents, including PMA, cycloheximide, AIF4, and okadaic acid. Although all of these agents stimulated c-Jun phosphorylation, cycloheximide and okadaic acid had no effect on pp42 MAP kinase phosphorylation, suggesting that MAP kinase activation was not necessary for c-Jun phosphorylation in vivo. Because dominant-negative RasAsn17 has been shown to block the effects of PMA on pp42 MAP kinase phosphorylation, we assessed its effect on c-Jun phosphorylation by cotransfection with a truncated c-Jun construct (c-Jun234). We...