Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs), p21Cip1/Waf1, p27Kip1 and p57Kip2, bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signalling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.

Inositol lipid phosphatases in membrane trafficking and human disease

The specific interaction of phosphoinositides with proteins is critical for a plethora of cellular processes, including cytoskeleton remodelling, mitogenic signalling, ion channel regulation and membrane traffic. The spatiotemporal restriction of different phosphoinositide species helps to define compartments within the cell, and this is particularly important for membrane trafficking within both the secretory and endocytic pathways. Phosphoinositide homoeostasis is tightly regulated by a large number of inositol kinases and phosphatases, which respectively phosphorylate and dephosphorylate distinct phosphoinositide species. Many of these enzymes have been implicated in regulating membrane trafficking and, accordingly, their dysregulation has been linked to a number of human diseases. In the present review, we focus on the inositol phosphatases, concentrating on their roles in membrane trafficking and the human diseases with which they have been associated.

Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras–GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras–GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.