14-3-3 Proteins Tune Non-Muscle Myosin-II Assembly, Providing a Possible Bridge between Cell Mechanics and Cancer Metastasis

Abstract

The 14-3-3 family comprises a group of small acidic regulatory proteins which are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3s sigma, epsilon, zeta, and eta correlate with a higher metastatic potential and poorer clinical outcomes in different cancers. We have uncovered a role for 14-3-3s in regulating the assembly of non-muscle myosin-II. This demonstrates that 14-3-3s could tune cell mechanics directly, and therefore contribute to the progression of metastatic cancers. Here, we examine how myosin-II assembly is regulated by 14-3-3 in Dictyostelium (one 14-3-3, one non-muscle myosin-II) and humans (seven 14-3-3s, three non-muscle myosin-IIs). In Dictyostelium, 14-3-3 mediates a pathway between microtubules and the racE small GTPase to regulate myosin-II assembly. Here, 14-3-3's expression levels negatively correlate with BTF accumulation. In vitro assembly assays using purified myosin-II tail fragments and 14-3-3 demonstrate that this interaction is direct, phosphorylation-independent, and high affinity (Kd ∼300 nM). We also found that the seven human paralogs of 14-3-3 affect the assembly of human non-muscle myosin-II filaments in different ways, some causing overassembly and others inhibiting assembly. These two 14-3-3 classes directly compete to govern the overall level of myosin-II assembly. Examining assembled myosin-II filaments by electron microscopy confirmed that the average filament size correlates with the overall assembly level. Furthermore, we mapped three critical residues which differ between the two 14-3-3 classes and discovered that alterations of any of these residues convert an assembler to a disassembler. Our findings demonstrate a novel phosphorylation-independent method for regulating myosin-II assembly that is mechanistically conserved from amoebas to humans. These findings imply that altered 14-3-3 expression profiles could directly modulate cell mechanics in metastatic cancers, which would be of great interest for basic and clinical sciences alike.