Molecular Mechanisms of Phospholipase C β3 Autoinhibition

Lyon, Angeline M.; Begley, Jessica; Manett, Taylor; Tesmer, John J.G.

doi:10.1016/j.str.2014.10.008

Cited by 40 publications

(82 citation statements)

References 55 publications

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“…1D) (45). Large CAT insertions in the phospholipase C isozymes play critical roles in regulating activity (46,47). We therefore sought to define the boundaries of the insertion and to assess its role in nSMase2 regulation.…”

Section: Resultsmentioning

confidence: 99%

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

Airola

Shanbhogue

Shamseddine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.sphingomyelinase | ceramide | enzyme | lipid | crystallography

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Section: Resultsmentioning

confidence: 99%

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

Airola

Shanbhogue

Shamseddine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…3AH8); and Gα q in complex with the regulator of G‐protein signaling 2 (RGS2) (Protein Data Bank accession no. 4EKC and 4QJ3) . The Gα q protein, which shares 90% identity at the amino acid level with Gα 11 , was used because crystal structures of Gα 11 are not available.…”

Section: Methodsmentioning

confidence: 99%

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss-of-Function Gα11 Mutation

Gorvin

Hannan

Cranston

et al. 2017

Journal of Bone and Mineral Research

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G‐protein subunit α‐11 (Gα11) couples the calcium‐sensing receptor (CaSR) to phospholipase C (PLC)‐mediated intracellular calcium (Ca2+ i) and mitogen‐activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss‐of‐function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild‐type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11, which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC‐mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR‐mediated Ca2+ i and extracellular signal‐regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss‐of‐function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR‐positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR‐mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss‐of‐function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

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“…The phosphorylated p‐loop of RAN was modeled based on the GTP‐bound crystal structure [Protein Data Bank (PDB; http://www.wwpdb.org/) ID: 4C0QJ (23). The phosphorylated p‐loop of RhoA is modeled on the GTPγSbound crystal structure (PDB ID: 1A2B) (24), and the phosphorylated p‐loop of G(α)q is modeled on the GDP‐AlF 4 ‐bound crystal structure (PDB ID: 4QJ5) (25).…”

Section: Methodsmentioning

confidence: 99%

LRRK2 autophosphorylation enhances its GTPase activity

Mobley

DeLucas

et al. 2015

FASEB j.

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The leucine-rich repeat kinase (LRRK)-2 protein contains nonoverlapping GTPase and kinase domains, and mutation in either domain can cause Parkinson disease. GTPase proteins are critical upstream modulators of many effector protein kinases. In LRRK2, this paradigm may be reversed, as the kinase domain phosphorylates its own GTPase domain. In this study, we found that the ameba LRRK2 ortholog ROCO4 phosphorylates the GTPase domain [termed Ras-of-complex (ROC) domain in this family] of human LRRK2 on the same residues as the human LRRK2 kinase. Phosphorylation of ROC enhances its rate of GTP hydrolysis [from k cat (catalytic constant) 0.007 to 0.016 min 21 ], without affecting GTP or GDP dissociation kinetics [k off = 0.093 and 0.148 min 21 for GTP and GDP, respectively). Phosphorylation also promotes the formation of ROC dimers, although GTPase activity appears to be equivalent between purified dimers and monomers. Modeling experiments show that phosphorylation induces conformational changes at the critical p-loop structure.Finally, ROC appears to be one of many GTPases phosphorylated in p-loop residues, as revealed by alignment of LRRK2 autophosphorylation sites with GTPases annotated in the phosphoproteome database. These results provide an example of a novel mechanism for kinase-mediated control of GTPase activity.-Liu, Z., Mobley, J. A., DeLucas, L. J., Kahn, R. A., West, A. B. LRRK2 autophosphorylation enhances its GTPase activity. FASEB J. 30, 336-347 (2016). www.fasebj.org

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Molecular Mechanisms of Phospholipase C β3 Autoinhibition

Cited by 40 publications

References 55 publications

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss-of-Function Gα11 Mutation

LRRK2 autophosphorylation enhances its GTPase activity

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