scholarly journals MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

2020 ◽  
Author(s):  
Ryan Ard ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Michael Phan ◽  
Radoslav Zinoviev ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Pdz Domain ◽  
Diacylglycerol Kinase ◽  
Binding Motif ◽  
Membrane Blebbing ◽  
Rhoa Activity ◽  
C Terminus ◽  
Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

scholarly journals Kalirin, a Key Player in Synapse Formation, Is Implicated in Human Diseases

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Prashant Mandela ◽  
Xin-Ming Ma
Keyword(s):  
Learning And Memory ◽  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Pdz Domain ◽  
Long Term Potentiation ◽  
Human Diseases ◽  
Binding Motif ◽  
Spine Density ◽  
Morphological Alterations

Synapse formation is considered to be crucial for learning and memory. Understanding the underlying molecular mechanisms of synapse formation is a key to understanding learning and memory. Kalirin-7, a major isoform of Kalirin in adult rodent brain, is an essential component of mature excitatory synapses. Kalirin-7 interacts with multiple PDZ-domain-containing proteins including PSD95, spinophilin, and GluR1 through its PDZ-binding motif. In cultured hippocampal/cortical neurons, overexpression of Kalirin-7 increases spine density and spine size whereas reduction of endogenous Kalirin-7 expression decreases synapse number, and spine density. In Kalirin-7 knockout mice, spine length, synapse number, and postsynaptic density (PSD) size are decreased in hippocampal CA1 pyramidal neurons; these morphological alterations are accompanied by a deficiency in long-term potentiation (LTP) and a decreased spontaneous excitatory postsynaptic current (sEPSC) frequency. Human Kalirin-7, also known as Duo or Huntingtin-associated protein-interacting protein (HAPIP), is equivalent to rat Kalirin-7. Recent studies show that Kalirin is relevant to many human diseases such as Huntington’s Disease, Alzheimer’s Disease, ischemic stroke, schizophrenia, depression, and cocaine addiction. This paper summarizes our recent understanding of Kalirin function.

scholarly journals NHE3 Regulatory Factor 1 (NHERF1) Modulates Intestinal Sodium-dependent Phosphate Transporter (NaPi-2b) Expression in Apical Microvilli

2012 ◽  
Vol 287 (42) ◽  
pp. 35047-35056 ◽  
Author(s):  
Hector Giral ◽  
DeeAnn Cranston ◽  
Luca Lanzano ◽  
Yupanqui Caldas ◽  
Eileen Sutherland ◽  
...  
Keyword(s):  
Small Intestine ◽  
Pdz Domain ◽  
Binding Motif ◽  
Rat Small Intestine ◽  
Regulatory Factor ◽  
Sequence Analyses ◽  
C Terminus ◽  
Expression Studies ◽  
Sodium Dependent ◽  
Rat Enterocytes

Pi uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary Pi but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2BBE cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1−/− mice, but not PDZK1−/− mice, had a diminished adaptation of NaPi-2b expression in response to a low Pi diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

scholarly journals Loquacious-PD facilitatesDrosophilaDicer-2 cleavage through interactions with the helicase domain and dsRNA

2017 ◽  
Vol 114 (38) ◽  
pp. E7939-E7948 ◽  
Author(s):  
Kyle D. Trettin ◽  
Niladri K. Sinha ◽  
Debra M. Eckert ◽  
Sarah E. Apple ◽  
Brenda L. Bass
Keyword(s):  
Amino Acids ◽  
Binding Motif ◽  
Helicase Domain ◽  
C Terminus ◽  
Dsrna Binding ◽  
Interaction Interface ◽  
Mechanistic Basis ◽  
Dsrna Cleavage

Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs inDrosophila. In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2’s helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.

scholarly journals Interactomic affinity profiling by holdup assay: Acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244613
Author(s):  
Pau Jané ◽  
Gergő Gógl ◽  
Camille Kostmann ◽  
Goran Bich ◽  
Virginie Girault ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Pdz Domain ◽  
Pi3k Pathway ◽  
Binding Motif ◽  
Pdz Domains ◽  
Post Translational Modifications ◽  
Binding Profile ◽  
Protein Motifs ◽  
C Terminus ◽  
The Impact

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

scholarly journals The C-terminal Tail of Presenilin Regulates Omi/HtrA2 Protease Activity

2004 ◽  
Vol 279 (44) ◽  
pp. 45844-45854 ◽  
Author(s):  
Sanjeev Gupta ◽  
Rajesh Singh ◽  
Pinaki Datta ◽  
ZhiJia Zhang ◽  
Christopher Orr ◽  
...  
Keyword(s):  
Cell Death ◽  
Proteolytic Activity ◽  
Protease Activity ◽  
Molecular Mechanisms ◽  
Pdz Domain ◽  
Ectopic Expression ◽  
Amyloid Β ◽  
Peptide Ligand ◽  
Binding Motif ◽  
Dependent Manner

Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease. Presenilins play an important role in amyloid β-precursor processing, NOTCH receptor signaling, and apoptosis. However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood. Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain. We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and β-casein and induces cell death in an Omi/HtrA2-dependent manner. Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death. Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis. This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins.

scholarly journals SHP-2 Positively Regulates Myogenesis by Coupling to the Rho GTPase Signaling Pathway

2004 ◽  
Vol 24 (12) ◽  
pp. 5340-5352 ◽  
Author(s):  
Maria I. Kontaridis ◽  
Seda Eminaga ◽  
Mara Fornaro ◽  
Christina Ivins Zito ◽  
Raffaella Sordella ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Intracellular Signaling ◽  
Rho Gtpase ◽  
Tyrosine Phosphatase ◽  
Signaling Cascades ◽  
Specific Gene ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Rho Family ◽  
Mechanistic Basis

ABSTRACT Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.

scholarly journals A unique insert in the genomes of high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1000 ◽  
Author(s):  
Noam Auslander ◽  
Yuri I. Wolf ◽  
Svetlana A. Shabalina ◽  
Eugene V. Koonin
Keyword(s):  
High Risk ◽  
Pdz Domain ◽  
Human Papillomaviruses ◽  
Machine Learning Techniques ◽  
Genome Comparison ◽  
Binding Motif ◽  
E6 Oncoprotein ◽  
C Terminus ◽  
Tumorigenic Potential ◽  
E6 And E7

The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6 as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.

scholarly journals Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

2021 ◽  
Author(s):  
Michael D Healy ◽  
Joanna Sacharz ◽  
Kerrie E McNally ◽  
Calum McConville ◽  
Ryan J Hall ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Pdz Domains ◽  
Sorting Nexin ◽  
Affinity Enrichment ◽  
C Terminus ◽  
Loop Sequence ◽  
Cargo Proteins

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

scholarly journals A unique insert in the genomes of high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1000 ◽  
Author(s):  
Noam Auslander ◽  
Yuri I. Wolf ◽  
Svetlana A. Shabalina ◽  
Eugene V. Koonin
Keyword(s):  
High Risk ◽  
Pdz Domain ◽  
Human Papillomaviruses ◽  
Machine Learning Techniques ◽  
Genome Comparison ◽  
Binding Motif ◽  
E6 Oncoprotein ◽  
C Terminus ◽  
Tumorigenic Potential ◽  
E6 And E7

The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6, as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that, as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.

scholarly journals Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

2012 ◽  
Vol 23 (20) ◽  
pp. 4008-4019 ◽  
Author(s):  
Ryan Ard ◽  
Kirk Mulatz ◽  
Hanan Abramovici ◽  
Jean-Christian Maillet ◽  
Alexandra Fottinger ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Critical Factor ◽  
Diacylglycerol Kinase ◽  
Rhoa Activity ◽  
Signaling Complex ◽  
Rhoa Activation ◽  
C Terminus ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Protein Kinase Cα ◽  
Underlying Mechanisms

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

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