Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Oancea, Elena; Teruel, Mary N.; Quest, Andrew F. G.; Meyer, Tobias

doi:10.1083/jcb.140.3.485

Cited by 320 publications

(256 citation statements)

References 45 publications

Supporting

Mentioning

242

Contrasting

Unclassified

Order By: Relevance

“…A small but functionally significant portion of RasGRP could be transiently recruited to the plasma membrane during the very brief period of diacylglycerol accumulation there following serum stimula- tion (42,48) and thus could be transiently brought in contact with plasma membrane-localized Ras GTPases. It should be noted that immunofluorescence studies of Sos1 and RasGRF-2 in serum-stimulated or ionophore-stimulated cells show predominant cytoplasmic distribution, with no detectable concentration at the plasma membrane (2,21), despite the evidence that these stimuli cause translocation of these GEFs to particulate fractions of the cell and stimulate GTP loading of Ras.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Tognon

Kirk

Passmore

et al. 1998

Molecular and Cellular Biology

221

207

View full text Add to dashboard Cite

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H-and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester-and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol-and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerolenriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

show abstract

Section: Discussionmentioning

confidence: 99%

“…phorbol ester (47,48). In addition to the histidines and cysteines required for zinc coordination, the C1 domain of mRas-GRP contains appropriate residues at the 11 positions that participate in forming a phorbol ester-binding pocket in the second C1 domain of PKC-␦ (64) (Fig.…”

Section: Selection Of a Cdna Clone Which Causes Ras-like Transformatimentioning

confidence: 99%

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Tognon

Kirk

Passmore

et al. 1998

Molecular and Cellular Biology

221

207

View full text Add to dashboard Cite

show abstract

“…Fig. 3C shows the increase in a 2 as a function of time (t) that was used to calculate the diffusion coefficient (slope ϭ 4D) of YFP-STIM1 under the basal condition (25). The average apparent diffusion coefficients of YFP-STIM1 were Ϸ0.1 and 0.05 m 2 /s before and after ER Ca 2ϩ depletion, respectively (Fig.…”

Section: A Mutant Stim1 That Lacks the C-terminal Polybasic Motif Sepmentioning

confidence: 99%

“…3B). This result indicates that the distance of recruitment for STIM1 puncta formation is Ͻ5 m. To determine the average distance over which STIM1 is recruited to ER-PM junctions, we measured the apparent diffusion coefficient (D) by fitting the two-dimensional relative fluorescence intensity profiles (I) to a Gaussian function, I ϭ exp (Ϫ(x 2 ϩ y 2 )/a 2 ) and calculating the increase in the square of the Gaussian peak radius (a 2 ) as YFP-STIM1 diffused back into the bleached area (25). Fig.…”

Section: A Mutant Stim1 That Lacks the C-terminal Polybasic Motif Sepmentioning

confidence: 99%

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca ²⁺ store depletion

Liou

Fivaz

Inoue

et al. 2007

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

Self Cite

573

695

View full text Add to dashboard Cite

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca 2؉ sensor that responds to ER Ca 2؉ store depletion and activates Ca 2؉ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca 2؉ store depletion. These oligomers rapidly dissociated when ER Ca 2؉ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca 2؉ store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 m to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca 2؉ gradients. Together, our live-cell measurements dissect the STIM1 ERto-PM signaling relay into four sequential steps: (i) dissociation of Ca 2؉ , (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca 2؉ channels.Ca 2ϩ release-activated Ca 2ϩ ͉ fluorescence recovery after photobleaching ͉ FRET ͉ store-operated Ca 2ϩ influx C a 2ϩ signals are used by cells for transducing receptor or electrical inputs into functional outputs such as gene expression, contraction, and secretion (1). Although endoplasmic reticulum (ER) Ca 2ϩ stores are the main source for inositol trisphosphate-induced transient Ca 2ϩ signals, a necessary step for activation of T lymphocytes, mast cells, and many other cells is a persistent increase in Ca 2ϩ concentration (2). These sustained Ca 2ϩ signals require retrograde signaling from the lumen of the ER to Ca 2ϩ channels in the plasma membrane (PM) in a process called store-operated Ca 2ϩ (SOC) influx [also termed Ca 2ϩ release-activated Ca 2ϩ (CRAC) in immune cells] (3). The Stauderman/Cahalan groups and our group have recently identified the single transmembrane protein stromal interaction molecule 1 (STIM1) as an ER Ca 2ϩ sensor that responds to the depletion of ER Ca 2ϩ and activates SOC/CRAC channels in the PM (4-6). STIM1 senses Ca 2ϩ by an EF hand Ca 2ϩ -binding site in the lumen of the ER (7). A recombinant fragment of the luminal region of STIM1 has been shown to form dimers and oligomers in the absence of Ca 2ϩ in vitro (8). Overexpression of STIM1 mutants with a disrupted EF hand Ca 2ϩ -binding motif resulted in constitutive activation of Ca 2ϩ influx (5, 6, 9). It was also observed that depletion of ER Ca 2ϩ stores induced STIM1 translocation to punctate structures near the PM that correspond to ER-PM junctions (5, 10). STIM1 colocalizes at these junctions with the CRAC channel Orai1 (also known as CRACM1) and synergistically activates SOC/CRAC infl...

show abstract

“…Upon stimulation, the enzyme adopts an open conformation whereby intramolecular interactions are interrupted and binding sites for intermolecular interactions that stabilize the open form are exposed, resulting in a catalytically active enzyme. In the case of PKC, these intermolecular interaction sites are the binding sites for phospholipids and anchoring proteins (27,28). We have previously suggested that one intramolecular interaction in ⑀PKC that maintains the enzyme in a closed form is between an ⑀RACK-binding site and a ⑀RACK site (17).…”

Section: Mathematical Modeling Of ⑀Pkc Translocation Suggests That ⑀Pmentioning

confidence: 99%

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Schechtman

Craske

Kheifets

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive ⑀PKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed⑀RACK. An amino acid difference between the ⑀RACK sequence in ⑀PKC and its homologous sequence in ⑀RACK constitutes a change from a polar non-charged amino acid (asparagine) in ⑀RACK to a polar charged amino acid (aspartate) in ⑀PKC. Here we show that mutating the aspartate to asparagine in ⑀PKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone-or PMAinduced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to ⑀RACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the ⑀RACK site and RACK-binding site within ⑀PKC is critical and rate limiting in the process of PKC translocation.The protein kinase C (PKC) 1 family of phospholipid (PL) -dependent serine/threonine kinases undergoes a conformational change and translocation, or movement, from the cytosolic to the cell particulate fraction upon activation (1, 2). Conformational changes in PKC from an inactive to an active state results in exposure of domains required for PKC anchoring to the particulate fraction and in increased sensitivity of the enzyme to proteases (1-3, 41). Therefore, the inactive state exists in a closed conformation, with the proteolytic sites protected, whereas the active state is in an open conformation with exposed proteolytic sites. Structural alterations from the closed to open states involve disruption of intramolecular interactions within the enzyme.An intramolecular interaction in inactive PKC between the catalytic site and a site in the regulatory domain that resembles a substrate phosphorylation site but lacks a serine or threonine phosphoacceptor (pseudosubstrate site) has been previously identified (3, 6). Deletion of the pseudosubstrate ( -substrate) site generated a constitutively active enzyme (6) and mutations of the basic residues in the -substrate site reduced the affinity of the catalytic site to the -substrate site generating a constitutively active enzyme, preferentially localized to the cell particulate fraction (6). Furthermore, conversion of the alanine in the -substrate site to a glutamic acid, mimicking a phosphorylated amino acid, resulted in loss of binding of the -substrate site to the catalytic site, creating a constitutively active enzyme (6). Finally, a peptide corresponding to the -substrate site is a competitive inhibitor of PKC catalytic activity (6).We previously demonstrated that translocation of PKC is associated with binding of each activated PKC isozyme to...

show abstract

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cited by 320 publications

References 45 publications

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca ²⁺ store depletion

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Contact Info

Product

Resources

About

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cited by 320 publications

References 45 publications

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca 2+ store depletion

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Contact Info

Product

Resources

About

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca ²⁺ store depletion