Reversible Phosphorylation as a Molecular Switch to Regulate Plasma Membrane Targeting of Acylated SH4 Domain Proteins

Abstract

Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes.Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated downregulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.

Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef

“…2A, indicated by white arrow). Consistent with previous findings (54), H(C5A)-and H(T6E)-Nef⌬SH4.GFP localized to intracellular compartments but also diffusely to the cytoplasm, as well as faintly to the PM (category 4, light gray bars in Fig. 2C).…”

“…4B) and did not reach its steady state distribution within the 3.5-h observation period, suggesting that in this case heterologous targeting routed Nef.GFP to a distinct anterograde transport pathway. Expectedly, the C5A and T6E Haspb SH4 domain mutants also mediated initial targeting to perinuclear membranes (54). However, these constructs accumulated in the cytoplasm and were not abundantly detected at the PM within the time frame of observation.…”

“…identical acylation pattern) and can substitute for that of Nef in mediating incorporation into HIV particles (39), Fyn and Yes were included because they mediate efficient PM association in their native context. The SH4 domain of the Leishmania Haspb protein mediates similarly efficient PM targeting; however, introduction of C5A and T6E mutations results in retargeting to the Golgi and/or perinuclear endosomes (54). In addition to these SH4 domain chimeras, the previously described Nef.GFP mutant PalmNef, which is palmitoylated because of a G3C mutation and thus enriched in detergent-resistant membranes relative to WT Nef (48,55), was included in our analysis.…”

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

“…These contained the N-terminal 18 amino acids of either Leishmania HASPB (Denny et al 2000;Stegmayer et al 2005) or of the mammalian SRC kinase YES1 (Tournaviti et al 2009) fused to GFP (SH4-HASPB-GFP) and mCherry (SH4-YES1-mCherry), respectively. Both fusion proteins are substrates for N-terminal myristoylation and subsequent palmitoylation (cysteine 5 in HASPB; cysteine 3 in YES1) (Supplemental Fig.…”

“…N-terminal SH4 domains are sufficient for intracellular targeting of, for example, GFP fusion proteins to the inner leaflet of plasma membranes (McCabe and Berthiaume 1999;Denny et al 2000;McCabe and Berthiaume 2001;Stegmayer et al 2005;Tournaviti et al 2007Tournaviti et al , 2009). Myristoylation of SH4 domains occurs cotranslationally and results in a transient insertion into intracellular membranes in a perinuclear region where they colocalize with Golgi markers (Wolven et al 1997;Denny et al 2000;Stegmayer et al 2005;Tournaviti et al 2007).…”