Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity

Czikora, Ágnes; Lundberg, Daniel J.; Abramovitz, Adelle; Lewin, Nancy E.; Kedei, Noémi; Peach, Megan L.; Zhou, Xiaoling; Merritt, Raymond C.; Craft, Elizabeth A.; Braun, Derek C.; Blumberg, Peter M.

doi:10.1074/jbc.m116.725333

Cited by 19 publications

(25 citation statements)

References 72 publications

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“…27 Additionally, Leu is present at this position in RasGRP2, a RasGRP isoform with very low phorbol ester binding affinity. 33 At this key homologous position, the switch between Trp and Tyr for the novel and conventional PKCs was found to influence their membrane affinity and cellular localization. 34 …”

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confidence: 99%

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: 99%

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

et al. 2018

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“…Like the DG75 B cell line, primary human B cells express RasGRP2, which however is not capable of connecting BCR engagement to activation of the Ras/Erk MAP kinase pathway. RasGRP2 possesses an unusual C1 domain that, unlike those of RasGRP1 and RasGRP3, does not bind DAG and hence is not regulated by the activity of PLCɣ 46 , 47 . Whether this lack of DAG responsiveness is responsible for the failure of RasGRP2 to connect BCR engagement to activation of the Erk MAP kinase pathway remains to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Grb2 and GRAP connect the B cell antigen receptor to Erk MAP kinase activation in human B cells

Vanshylla

Bartsch

Hitzing

et al. 2018

Sci Rep

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The B cell antigen receptor (BCR) employs enzymatically inactive adaptor proteins to facilitate activation of intracellular signaling pathways. In animal model systems, adaptor proteins of the growth factor receptor-bound 2 (Grb2) family have been shown to serve critical functions in lymphocytes. However, the roles of Grb2 and the Grb2-related adaptor protein (GRAP) in human B lymphocytes remain unclear. Using TALEN-mediated gene targeting, we show that in human B cells Grb2 and GRAP amplify signaling by the immunoglobulin tail tyrosine (ITT) motif of mIgE-containing BCRs and furthermore connect immunoreceptor tyrosine-based activation motif (ITAM) signaling to activation of the Ras-controlled Erk MAP kinase pathway. In contrast to mouse B cells, BCR-induced activation of Erk in human B cells is largely independent of phospholipase C-ɣ activity and diacylglycerol-responsive members of Ras guanine nucleotide releasing proteins. Together, our results demonstrate that Grb2 family adaptors are critical regulators of ITAM and ITT signaling in naïve and IgE-switched human B cells.

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“…Although classified as a “typical” C1 domain, the C1a domain of PKCθ was described as having an affinity for PDBu of >200 nM [43] and an affinity for DAG of 2000 nM, compared to 26 nM for the PKCθ C1b domain [44], and it did not contribute to PKCθ translocation (unpublished observations). Although classified as “atypical”, the C1 domain of RasGRP2 in fact bound PDBu with an affinity of 2,900 nM [45]. …”

Section: Discussionmentioning

confidence: 99%

The C1 domain of Vav3, a novel potential therapeutic target

Kelsey

Géczy

Kaler

et al. 2017

Cellular Signalling

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Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.

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Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity

Cited by 19 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

Grb2 and GRAP connect the B cell antigen receptor to Erk MAP kinase activation in human B cells

The C1 domain of Vav3, a novel potential therapeutic target

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