Abstract
The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.
Highlights
The activity of PKC3 is critical to many signaling pathways, through which protein kinase C (PKC) elicits a variety of physiological outputs [1, 2]
Whereas PKC may be constitutively activated through proteolysis, the principal mechanism for the activation of the conventional (␣, the alternatively spliced ⌱ and ⌱⌱, ␥) and novel (␦, ⑀, , ) PKC isozymes is via translocation of PKC to the membrane; this translocation is driven by the C1 domain of these PKC isozymes, which binds diacylglycerol (DG) or the functional analogues, phorbol esters [3]
We chose the C1b domain of the conventional isoform PKCI/II because the isolated domain is stable and amenable to biochemical studies [5, 16, 17, 29]; it is part of a conventional PKC isoform whose biochemical and biophysical properties have been well studied (3, 5, 12, 13, 17, 29 –31). (This domain will hereafter be referred to as C1b, as the C1b domain is identical in both isoforms.) This C1b domain does not contain an endogenous Trp fluorophore, we introduced Trp at four positions in the domain: F114W, H117W, Y123W, and L150W (PKCII numbering)
Summary
The activity of PKC3 is critical to many signaling pathways, through which PKC elicits a variety of physiological outputs [1, 2]. We have recently shown that a single amino acid in the C1 domain (at position 22) tunes the affinity of this domain for DG-containing membranes [4] This residue lies on the outside face of one of the sides of the ligand binding pocket. When present as Trp, as it is in novel PKC isozymes, the domain binds DG-containing membranes with sufficiently high affinity to sense agonist-evoked generation of DG. When present as Tyr, as it is in conventional isozymes, the affinity for DG-containing membranes is an order of magnitude lower, requiring a second targeting mechanism (Ca2ϩ-dependent engagement of the C2 domain) for translocation to membranes. We take advantage of engineering a Trp at the tuning position of a conventional PKC to obtain a C1 domain that has a high membrane affinity and a built-in fluorophore
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