Protein kinase C μ is negatively regulated by 14-3-3 signal transduction proteins

Abstract

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3τ isoform has been shown to associate with protein kinase C θ and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3τ interacts with protein kinase C μ (PKCμ), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCμ and 14-3-3τ can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCμ-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCμ deletion mutants, the 14-3-3τ binding region is mapped within the regulatory C1 domain. Binding of 14-3-3τ to PKCμ is significantly enhanced upon phorbol ester stimulation of PKCμ kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3τ is not a substrate of PKCμ. In contrast 14-3-3τ strongly down-regulates PKCμ kinase activity in vitro. Moreover, overexpression of 14-3-3τ significantly reduced phorbol ester induced activation of PKCμ kinase activity in intact cells. We therefore conclude that 14-3-3τ is a negative regulator of PKCμ in T cells.