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.

Protein kinase D (PKD) regulates the fission of vesicles from the trans-Golgi-network 1,2 . We show that phosphatidylinositol-4-kinase III beta (PI4KIIIβ), a key player in Golgi complex structure and function 3 , is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIβ at a motif highly conserved from yeast to man. PKD mediated phosphorylation stimulated lipid kinase activity of PI4KIIIβ and enhanced VSV-G transport to plasma membrane. The identification of PI4KIIIβ as one of the PKD substrates should help to reveal the molecular events leading to transport carrier formation.

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase C␥-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.The mechanistic elucidation of signaling pathways regulating dynamic actin remodeling processes in migrating cells is pivotal to a comprehensive understanding of cancer cell metastasis. Protein kinase D (PKD) 2 has recently been identified as a vital upstream regulator of polarized cell motility and F-actin organization (1-4). PKD localizes to sites of dynamic actin remodeling (1). The kinase activity is essential for the control of directed cell motility (1, 2), whereby active PKD1 inhibited, whereas kinase-inactive PKD1KD strongly enhanced motility and invasiveness (1-4). Mechanistically, a key role for PKD1 in controlling the activity of the ubiquitous F-actin depolymerizing-and severing factor cofilin via slingshot1L (SSH1L) cofilin phosphatase has been demonstrated. The activity of SSH1L is mainly regulated by its binding to filamentous actin (F-actin), which has been shown to strongly enhance its activity (5, 6). Phosphorylation of SSH1L at Ser 978 by active PKD1, e.g. downstream of RhoA or oxidative stress, generates a 14-3-3 binding motif within an important F-actin binding region, thus resulting in the sequestration of SSH1L away from dynamic actin structures, reducing SSH1 activity and active non-S3-phosphorylated cofilin levels (2). By severing actin filaments, cofilin increases both the availability of G-actin monomers as well as the number of "barbed ends" for polymerization (7). Furthermore, severed filaments are the preferred substrate for dendritic nucleation by the Arp complex (8, 9). Localized induction of actin polymerization and the formation of branched actin networks constitute the basis for membrane protrusion and cell motility (2, 10, 11). In line with an upstream regulatory role in the control of these processes, PKD1 and -2 are also capable of binding to F-actin in vitro (1). In the case of PKD1, in vivo F-actin binding has been demonstrated as well, which most likely facilitates an interaction with actin regulatory proteins such as SSH1L (2). We now have identified a second key regulatory signaling pathway ...

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L. [Cancer Res 2009;69(14):5634-8]

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-B activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-B activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/ CD40 ligand-induced NF-B activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2) 2 -TRAF1 versus TRAF2-(TRAF1) 2 ) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling. CD40 and its ligand CD40L1 /CD154 are members of the tumor necrosis factor (TNF) receptor and TNF ligand family and represent major regulators of lymphocyte function (1). Aside from T-and B-cells, CD40 and CD40L are expressed in a variety of non-lymphocytic cell types including monocytes, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (1). The CD40/CD40L system plays a critical role in the regulation of thymus-dependent humoral immune responses but also contributes to chronic inflammatory processes in autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis (1).Engagement of CD40 results in the recruitment of members of the TNF receptor-associated factor (TRAF) adaptor protein family (1, 2). In addition, triggering of CD40 leads to Janus family kinase 3 (Jak3)-dependent activation of signal transducers and activators of transcription (STAT) proteins and to activation of the Src-related tyrosine kinase Lyn (3-6). TRAF proteins couple TNF receptors and Toll/interleukin-1 receptor family members to pathways leading to the activation of the inhibitor of I-B kinases and kinases of the mitogen-activated protein kinase (MAPK) family (2). All members of the TRAF family share a conserved C-terminal homology domain of ϳ180 amino acids (TRAF domain), which mediates interactions with the above mentioned receptors and the majority of cytosolic factors known for their TRAF binding capacity, including kinases, inhibitor of apoptosis proteins, and death domain adaptor proteins (2). With the exception of TRAF1, the N-terminal domain of all six known mammalian TRAFs comprise a single RING finger followed by several zinc finger motifs (2) that are important for ...

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC partially colocalizes with PKC in different cell types. Colocalization depends on the presence of the PKC pleckstrin homology domain. Coexpression of constitutively active PKC with PKC leads to a significant enhancement of the PKC substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser 738/742 of PKC, whereas Ser 910 autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC directly phosphorylates PKC on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC/PKC affecting different arms of the p38/ERK/ JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC for interaction with and activation by PKC as pleckstrin homology domain and activation loop.