Identification of the activator-binding residues in the second cysteine-rich regulatory domain of protein kinase Cθ (PKCθ)

Rahman, Ghazi Muhammad Sayedur; Shanker, Sreejesh; Lewin, Nancy E.; Kedei, Noémi; Hill, Colin; Prasad, B. V. Venkataram; Blumberg, Peter M.; Das, Joydip

doi:10.1042/bj20121307

Cited by 25 publications

(29 citation statements)

References 72 publications

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“…The NMR structure of Munc13-1 also revealed that the side chain of Trp-22 in the C1 domain (Trp-588 in the full-length Munc13-1) occludes the ligand binding site 30 (Figure 1). The orientation of the tryptophan residue at the homologous position in the C1 domain of PKC δ 31 and PKC θ 32 is different than that of Munc13-1. Most of the phorbol ester-sensitive C1 domains contain Trp at this site with few exceptions (Tyr in C1B of PKC( α / β / γ ); Val in MRCK α / β ; Leu in MRCK γ ; and Phe in PKD2C1A.…”

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confidence: 83%

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation

et al. 2018

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Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ;. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

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confidence: 83%

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation

et al. 2018

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“…85–87 Only C1B from PKCδ proved to be crystallizable when complexed to the following ligands: a water-soluble phorbol ester, phorbol-13 acetate; 85 and general anesthetics, methoxymethylcyclopropane and cyclopropylmethanol that bind to the surface of the domain outside of the canonical DAG pocket. 86,87 There are currently no structures of C1 complexed to its native PKC agonist, diacylglycerol.…”

Section: Individual Domains Of Pkcmentioning

confidence: 99%

Dynamics and Membrane Interactions of Protein Kinase C

Igumenova

2015

Biochemistry

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Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic auto-inhibited latent form to the membrane-associated active form. The membrane recruitment step is accompanied by the conformational rearrangement of the enzyme, which relieves auto-inhibitory interactions and thereby enables PKC to phosphorylate its targets. The multi-domain structure and intrinsic flexibility of PKC present remarkable challenges and opportunities for the biophysical and structural biology studies of this class of enzymes and their interactions with membranes – the major focus of this Current Topics article. I will highlight the recent advances in the field, outline the current challenges, and identify areas where biophysics and structural biology approaches can provide insight into the isoenzyme-specific regulation of PKC activity.

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“…In animals, DAG serves as a classical second messenger that activates protein kinase C (PKC) by binding to its C1 domain [153]. However, no homologous PKC genes have been identified in plants.…”

Section: Diacylglycerolmentioning

confidence: 99%

Plant phosphoinositide-dependent phospholipases C: Variations around a canonical theme

et al. 2014

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Phosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca(2+)-dependent manner, phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) into diacylglycerol (DAG) and inositol triphosphate (IP3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCζs, the simplest animal PI-PLCs. Like these animal counterparts, they are only composed of EF-hand, X/Y and C2 domains. However, plant PI-PLCs do not have a conventional EF-hand domain since they are often truncated, while some PI-PLCs have no EF-hand domain at all. Despite this simple structure, plant PI-PLCs are involved in many essential plant processes, either associated with development or in response to environmental stresses. The action of PI-PLCs relies on the mediators they produce. In plants, IP3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) also transmit signals, thus highlighting the importance of coupling PI-PLC action with inositol-phosphate kinases and phosphatases. PI-PLCs also produce a lipid molecule, but plant PI-PLC pathways show a peculiarity in that the active lipid does not appear to be DAG but its phosphorylated form, phosphatidic acid (PA). Besides, PI-PLCs can also act by altering their substrate levels. Taken together, plant PI-PLCs show functional differences when compared to their animal counterparts. However, they act on similar general signalling pathways including calcium homeostasis and cell phosphoproteome. Several important questions remain unanswered. The cross-talk between the soluble and lipid mediators generated by plant PI-PLCs is not understood and how the coupling between PI-PLCs and inositol-kinases or DAG-kinases is carried out remains to be established.

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Identification of the activator-binding residues in the second cysteine-rich regulatory domain of protein kinase Cθ (PKCθ)

Cited by 25 publications

References 72 publications

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation

Dynamics and Membrane Interactions of Protein Kinase C

Plant phosphoinositide-dependent phospholipases C: Variations around a canonical theme

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