Role of T-loop Phosphorylation in PDK1 Activation, Stability, and Substrate Binding

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

PDZ domains1 are modular protein interaction domains that play a role in protein targeting and protein complex assembly. Once termed Discs-large homology regions (DHRs) or GLGF repeats (after a conserved Gly-Leu-Gly-Phe sequence found within the domain), these domains of ∼90 amino acids are now primarily known by an acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, theDrosophila septate junction protein Discs-large, and the tight junction protein ZO-1.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

ADP-ribosylation actor 6 (ARF6) regulates the endocytosis and recycling of a variety of proteins and also promotes peripheral actin rearrangements and cell motility. ARF6 is activated by a large number of guanine nucleotide exchange factors, which likely regulate ARF6 at different locations and during different processes. In this study we investigate the roles of the cytohesin ADP-ribosylation factor (ARF)-guanine nucleotide exchange factors during the recycling of integrin beta1. Intriguingly, we find that knockdown and overexpression of ARNO/cytohesin 2 and GRP1/cytohesin 3 have opposing effects on cell adhesion and spreading on fibronectin and on cell migration. We find that ARNO/cytohesin 2 is required for integrin beta1 recycling, whereas GRP1/cytohesin 3 is dispensable for this process. This is the first demonstration of unique roles for these proteins.

Synaptic AMPA Receptor Plasticity and Behavior

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations

Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.

Transforming growth factor-beta-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain alphaG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic alphaG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of alphaG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.

Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.

Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1, acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases