Structure of the C2 domain from novel protein kinase Cϵ. A membrane binding model for Ca2+-independent C2 domains

Ochoa, Wendy F.; Garcı́a-Garcı́a, Josefa; Fita, Ignacio; Corbalán-Garcı́a, Senena; Verdaguer, N.; Gómez‐Fernández, Juan C.

doi:10.1006/jmbi.2001.4910

Cited by 96 publications

(104 citation statements)

References 41 publications

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“…Since the steady state level of D86A in the particulate fraction was similar to the steady state level of the wild-type enzyme, and the second step of translocation (binding to the membrane) was the same for all mutants, the rate of closing of an enzyme, once it was open, could be considered negligible. Ochoa et al (29) demonstrated that the binding of the V1 domain to PL is not altered by D86A mutation, supporting our hypothesis that the second step of translocation (binding to the membrane) is not altered. However, for the N mutant, the k 1 (rate of ⑀PKC opening) was slower than that of D86A and similar to wild-type ⑀PKC, and the k Ϫ1 (rate of ⑀PKC closing) was no longer negligible.…”

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

confidence: 89%

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Schechtman

Craske

Kheifets

et al. 2004

Journal of Biological Chemistry

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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...

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

confidence: 89%

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Schechtman

Craske

Kheifets

et al. 2004

Journal of Biological Chemistry

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“…The components located at 1662 (15%) and 1686 cm −1 (4%) arise from β-turns and the 1674 cm −1 band (9%) is usually assigned to antiparallel β-sheet structure [3,32,33,52]. Quantitation of the secondary structure and assignments of the PKCε-C2 domain in D 2 O are also summarized in Table 4 A very good agreement was also reached between the secondary structure deduced from infrared spectroscopy and that concluded by means of X-ray diffraction from a crystal of this protein [55]. Table 2 shows that for β-pleated sheet the percentages found were 60% for infrared spectroscopy and 54% for X-ray diffraction (XRD), for α-helix 21% (IR) and 20% (XRD) and for β-turns 19% (IR) and 20% (XRD).…”

Section: Structure Of the Pkcε-c2 Domainsupporting

confidence: 60%

Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

Corbalán-Garcı́a

Garcı́a-Garcı́a

Sánchez-Carrillo

et al. 2003

Journal of Spectroscopy

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Abstract. The amide I regions in the original infrared spectra of PKCα-C2 in the Ca 2+ -free and Ca 2+ -bound states are both consistent with a predominantly β-sheet secondary structure. Spectroscopic studies of the thermal denaturation revealed that for the PKCα-C2 domain alone the secondary structure abruptly changed at 50 • C. While in the presence of Ca 2+ , the thermal stability of the protein increased considerably. Phosphatidic acid binding to the PKCα-C2 domain was characterized, and the lipid-protein binding becoming Ca 2+ -independent when 100 mol% phosphatidic acid vesicles was used. The effect of lipid binding on secondary structure and thermal stability was also studied. In addition, the secondary structure of the C2 domain from the novel PKCε was also determined by IR spectroscopy and β-sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I band starting at 45 • C. Phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure. It was observed through thermal stability experiments that the secondary structure did not change upon lipid binding and the protein stability was very high with no significant changes occurring in the secondary structure after heating.

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“…The N-terminus of PKCe contains a C2-like domain that may be important for its intracellular targeting and interactions with other proteins (Schechtman and Mochly-Rosen, 2001). However, crystal structures of the PKCe-C2 domain (residues Met 1 to Gly 138 ) consist of an eight-stranded, antiparallel, b-sandwich (Ochoa et al, 2001) that would not be suited for helical packing/bundling with a protein such as Bax. PKCe does not contain a BH3-like domain and, until the complete crystal structure of PKCe has been resolved, it is not possible to predict whether the surface of this protein presents a suitable interface for interacting directly with Bax protein.…”

Section: Discussionmentioning

confidence: 99%