Search

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC)[micro] involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC[micro]--green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the N[H.sub.2]-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC[micro] completely abrogated Golgi localization of PKC[micro]. As an N[H.sub.2]-terminal PKC[micro] fragment was colocalized with p24, this region of PKC[micro] is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC[micro] to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinasedead PKC[micro] found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC[micro], in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of N[H.sub.2]-terminal serine(s) in the regulatory domain. PKC[micro] activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC[micro] function at the Golgi compartment.

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 (1-3) but that also has endocrine (4,5) and enzymatic functions (6,7). 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 (8,9) that also promotes degradation of the cyclin-dependent kinase inhibitor [p27.sup.Kip1] (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 NF[kappa]B. 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 [p27.sup.Kip1] expression through stabilization of [p27.sup.Kip1] protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys60 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., There is evidence for intracellular functions of MIF (1,4,6,7), and we thought that these intracellular functions might be based on direct interactions of MIF with intracellular proteins. To identify a [...]

Protein kinase D3 (PKD3) is upregulated in triple‐negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro‐oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA‐MB‐231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF‐H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF‐H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC. What's new? The serine‐threonine kinase PKD3 is upregulated in triple‐negative breast cancer (TNBC) and associated with cell proliferation and metastasis. The mechanisms underlying PKD3 pro‐oncogenic function remain unclear, however. Here, the authors reveal a critical role for PKD3 in the maintenance and propagation of TNBC stem cells in vitro and in vivo. The function of PKD3 in the maintenance of cancer stem cells requires upstream activation by GEF‐H1. Importantly, the PKD inhibitor CRT0066101 and paclitaxel synergistically decrease TNBC stem cell prevalence and tumor recurrence in vivo. The results provide a rationale for targeting PKD3 to eliminate the tumor‐initiating cell population in TNBC. [ABSTRACT FROM AUTHOR]

Synaptic connections in the brain are continuously weakened or strengthened in response to changes in neuronal activity. This process, known as synaptic plasticity, is the cellular basis for learning and memory, and is thought to be altered in several neuronal disorders. An important aspect of synaptic plasticity is the tightly controlled trafficking and synaptic targeting of the AMPA-type glutamate receptors, which are the major mediators of fast excitatory transmission in the brain. This review addresses the role of Rab GTPases in AMPA receptor trafficking in neurons under basal conditions and during activity-induced synaptic plasticity, especially during long-term potentiation (LTP) and long-term depression (LTD). We highlight the importance of the tight spatio-temporal control of Rab activity and suggest that this is critical for proper neuronal functions. We also discuss how abnormal AMPA receptor trafficking and malfunctioning of Rabs can lead to neurologic disorders or memory problems. [ABSTRACT FROM AUTHOR]