Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity.

p53 is a transcription factor that mediates tumor suppressor responses. Correct folding of the p53 protein is essential for these activities, and point mutations that induce conformational instability of p53 are frequently found in cancers. These mutant p53s not only lose wild-type activity but can also acquire the ability to promote invasion and metastasis. We show that folding of wild-type p53 is promoted by an interaction with the chaperonin CCT. Depletion of this chaperone in cells results in the accumulation of misfolded p53, leading to a reduction in p53-dependent gene expression. Intriguingly, p53 proteins mutated to prevent the interaction with CCT show conformational instability and acquire an ability to promote invasion and random motility that is similar to the activity of tumor-derived p53 mutants. Our data therefore suggest that both growth suppression and cell invasion may be differentially regulated functions of wild-type p53.

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways.

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.

Costimulation of dectin-1 and DC-SIGN triggers the arachidonic acid cascade in human monocyte-derived dendritic cells.

Inflammatory mediators derived from arachidonic acid (AA) alter the function of dendritic cells (DC), but data regarding their biosynthesis resulting from stimulation of opsonic and nonopsonic receptors are scarce. To address this issue, the production of eicosanoids by human monocyte-derived DC stimulated via receptors involved in Ag recognition was assessed. Activation of FcgammaR induced AA release, short-term, low-grade PG biosynthesis, and IL-10 production, whereas zymosan, which contains ligands of both the mannose receptor and the human beta-glucan receptor dectin-1, induced a wider set of responses including cyclooxygenase 2 induction and biosynthesis of leukotriene C(4) and IL-12p70. The cytosolic phospholipase A(2) inhibitor pyrrolidine 1 completely inhibited AA release stimulated via all receptors, whereas the spleen tyrosine kinase (Syk) inhibitors piceatannol and R406 fully blocked AA release in response to immune complexes, but only partially blocked the effect of zymosan. Furthermore, anti-dectin-1 mAb partially inhibited the response to zymosan, and this inhibition was enhanced by mAb against DC-specific ICAM-3-grabbing nonintegrin (SIGN). Immunoprecipitation of DC lysates showed coimmunoprecipitation of DC-SIGN and dectin-1, which was confirmed using Myc-dectin-1 and DC-SIGN constructs in HEK293 cells. These data reveal a robust metabolism of AA in human DC stimulated through both opsonic and nonopsonic receptors. The FcgammaR route depends on the ITAM/Syk/cytosolic phospholipase A(2) axis, whereas the response to zymosan involves the interaction with the C-type lectin receptors dectin-1 and DC-SIGN. These findings help explain the distinct functional properties of DC matured by immune complexes vs those matured by beta-glucans.

Coupling of C3bi to IgG inhibits the tyrosine phosphorylation signaling cascade downstream Syk and reduces cytokine induction in monocytes.

The effect of coupling C3bi to immunoglobulin G (IgG) immune complexes (IC) on their ability to produce protein tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and the Akt/protein kinase B (PKB) routes was assessed in human monocytes. Cross-linking Fc receptors for IgG activated the protein tyrosine kinase Syk, phospholipases Cgamma1 and Cgamma2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the gamma-chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their beta-glucan content mimic the effect of fungi, produced a limited increase of tyrosine-phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2), interleukin (IL)-1beta, and eotaxin-2/CCL24, which were not observed with C3bi-coated IC. Zymosan induced the expression of tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-10, IL-6, and MCP-2/CCL8, whereas the cytokine signature of C3bi-coated zymosan also included interferon-inducible protein 10/CXC chemokine ligand 10, platelet-derived growth factor-BB, and I-309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.