Increased Membrane Affinity of the C1 Domain of Protein Kinase Cδ Compensates for the Lack of Involvement of Its C2 Domain in Membrane Recruitment

Abstract

Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membranetargeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca 2؉ -triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKC␦. We show that PKC␦ and a conventional isozyme, PKC␤II, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKC␤II with membranes, the C2 domain of PKC␦ does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKC␦ with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKC␦ in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKC␦. Thus, the increased affinity of the C1 domain of PKC␦ allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca 2؉ binding to the C2 domain and DAG binding to the C1 domain for activation.Many signaling proteins translocate from the cytosol to the membrane to become activated or to reach their substrates. Reversible binding to the membrane is mediated by modules such as the C1, C2, pleckstrin homology, and FYVE domains, with each recognizing specific lipid determinants or membrane properties. The protein kinase C (PKC) 3 family is a classic example of an enzyme whose function is regulated by membrane translocation. For this family of signaling proteins, the C1 and C2 domains direct the enzyme to the membrane following generation of diacylglycerol (DAG) and, for some isoforms, CaThe C1 domain is a 5-kDa globular domain composed primarily of two ␤-sheets that are pulled apart to expose a binding pocket for DAG and phorbol. Found in a number of other proteins in addition to PKC (notably, protein kinase D, DAG kinase, and Raf), this domain is an effective DAG sensor when expressed alone (reviewed in Refs. 2 and 3). Moreover, the C1 domain selectively recognizes phosphatidylserine (PS) (4, 5). The C2 domain is another small (16 kDa) globular domain that is primarily composed of ␤-strands; in this case, three loops in the ␤-strands at one end of the domain form a Ca 2ϩ -binding site. Conventional C2 domains such as those found in phospholipase C, phospholipase A 2 , phosphatidylinositol 3-kinase, synaptotagmin, and Munc13 bind Ca 2ϩ in this pocket, and the binding drives the membrane interaction (reviewed in Refs. 6 and 7). Novel C2 domains such as that found in PTEN do not bind Ca 2ϩ , yet can still bind phospholipids (8). The coordinated use of a C1 and C2 domain allows sensitivity in the...

Isoform Specificity of PKC Translocation in LivingAplysiaSensory Neurons and a Role for Ca2+-Dependent PKC APL I in the Induction of Intermediate-Term Facilitation

“…The better translocation of Apl II by DOG alone compared with Apl I could be attributable to a higher affinity of the C1 domain of Apl II for DOG, as has been shown for Ca 2ϩ -independent PKCs in vertebrates (Giorgione et al, 2006). Although the C1 domains of Apl I and Apl II bind phorbol esters to a similar extent , this does not necessarily predict affinities for DOG (Slater et al, 1996).…”

Lipid activation of protein kinases

“…Diacylglycerol levels at the Golgi are significantly elevated compared with the plasma membrane under basal conditions, and, in addition, agonist-evoked increases of this lipid second messenger are much more sustained at the Golgi compared with the plasma membrane. The unique profile of diacylglycerol at Golgi produces, in turn, a PKC signature unique to Golgi; not only is there preferential recruitment of novel PKCs, which have an intrinsically higher affinity for diacylglycerol because of a C1 domain tuned for tighter binding to diacylglycerol (34,35), but the agonist-evoked activity at Golgi is much more prolonged than at the plasma membrane (33).…”

Protein kinase C as a stress sensor

“…In addition, novel PKCs have C2 domains which do not bind to known ligands, and,thus, are not activated by calcium signals. It is thought that without the calcium sensor, novel PKCs translocate and activate differently than conventional PKCs [6]. PKCε is a member of this group.…”