Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.
Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C tt (PKC~t) in vitro and in vivo. Among the staurosporine-derived, rather selective PKC inhibitors the indolocarbazole G6 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKC~t with an IC5o of 20 nM, whereas the bisindolylmaleimide G6 6983 was extremely ineffective in suppressing PKCtt kinase activity with a thousand-fold higher ICso of 20 ~M. Other strong inhibitors of PKC~t were the rather unspecific inldbitors staurosporine and K252a. Contrary to the poor inhibition of PKCtt by G~ 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC5o values from 7 to 60 nM. Thus, Gii 6983 was able to differentiate between PKC~t and other PKC isoenzymes being useful for selective determination of PKC~t kinase activity in the presence of other PKC isoenzymes.
Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (␣, I, II, ␥, ␦, ⑀, , , and ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position ؉1 carboxyl-terminal of the phosphorylated Ser and basic residues at position ؊3. All isozymes, except PKC, selected peptides with basic amino acids at positions ؊6, ؊4, and ؊2. PKC␣, -I, -II, -␥, and -selected peptides with basic amino acid at positions ؉2, ؉3, and ؉4, but PKC␦, -⑀, -, and -preferred peptides with hydrophobic amino acid at these positions. At position ؊5, the selectivity was quite different among the various isozymes; PKC␣, -␥, and -␦ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position ؊3 to ؉2 for PKC␣, -I, -II, -␥, -␦, and -were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC␣, -I, -␦, -, and -were prepared and used for the determination of K m and V max for these isozymes. As judged by V max /K m values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft. Protein kinase C (PKC)1 family members play crucial roles in the signal transduction of a variety of extracellular stimuli, such as hormones and growth factors (1). To date, twelve isozymes of PKC have been identified in mammalian tissues and subdivided into conventional PKC (cPKC) members comprising ␣, I, II, and ␥ isoforms (activated by calcium, acidic phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising ␦, ⑀, , and (activated by DAG and acidic phospholipid but insensitive to calcium), and atypical PKCs (aPKC) / and (mechanism of regulation not clear) (1-6). Another subgroup of PKCs may be defined by PKC, which has a potential signal peptide and transmembrane domain (7). Since these PKC isozymes differ in their expression in different tissues and in their mode of activation (1), each isozyme may play some specific role in signal transduction processes. Recent investigations using various approaches such as overexpression and down-regulation of specific isozymes support this idea (1,5). A large number of proteins have been shown to be phosphorylated by PKC in ...
In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKCp, specific antibodies have been raised against unique aminoand carboxy-terminal regions. PKCp kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogeneous PKCp gene. Cell fractionation revealed that PKCp is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane-bound structures. In vitro kinase assays with immunoprecipitated PKCp demonstrated a Ca2+ independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limited in vitro phorbol ester response, an apparent phorbol ester activation of PKCp was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12-myristate 13-acetate or 1,2 dioleoyl-sn-glycerol. Both in vitro autophosphorylation and substrate phosphorylation of myelin basic protein and histone I11 were enhanced under these conditions. However, long-term treatment with the phorbol ester did not result in downregulation of PKCp protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKCp, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60-fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKCp and regulation of its kinase activity differ from that of other PKCs suggesting a novel function of PKCp in intracellular signal pathways.Keywords. Protein kinase Cp; activators, of protein kinase Cp; inhibitors of protein kinase Cp.Protein kinases C (PKC) are important regulatory enzymes involved in multiple cellular responses. They are typically activated by lipid second messengers such as diacylglycerol in response to various extracellular agonists like hormones, neurotransmitters, growth factors and cytokines [ l , 21. Molecular cloning of various PKC isoforms has established that PKC is a multigene family [3 -111. All isozymes share a characteristic conserved catalytic domain in the carboxy-terminal region and an amino-terminal regulatory site (Cl). To date, 11 members have been identified, which can be grouped into three major classes according to their different activation conditions. The first group, the conventional PKCs (cPKCa, p l , p2 and y ) require Ca2' to be activated in the presence of phosphatidylserine [3] whereas the second group, the novel PKCs (nPKCs) lack the C2 domain of cPKCs and are Ca2+ independent [4-6, 8, 91. Common features within the C l domain of cPKCs and nPKCs are a conserved pseudosubstrate site and two adjacent aminoterminal cysteine clusters that are involved in phorbol ester binding [2]. Members of the third subgroup have been termed atypical PKCs (aPKCC, and A) because they lack the C2 region and contain o...
Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12] [13] and Fas-associated DD protein (FADD/MORT1) [14] [15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17] [18] [19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13] [20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7] [8] [9] [10] [21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-DeltaFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-DeltaFADD did not interfere with TNF-mediated gene induction or with activation of NF-kappaB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-DeltaFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.
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