Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

Abstract

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1␥) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1␥ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1⌬DAD), indicating that deregulation of autoinhibition is effective in FMNL1␥. Expression of both FMNL1␥ and FMNL1⌬DAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.Formins represent a protein family indispensable for many fundamental actin-dependent processes, including migration, vesicle trafficking, morphogenesis, and cytokinesis (1). Because these polarized processes are also involved in inflammation, deregulated proliferation, and metastasis, formins have been suggested to represent attractive drug targets for inflammatory and malignant diseases. Formin-like 1 (FMNL1) 3 is expressed restrictedly in hematopoietic lineagederived cells and overexpressed in malignant cells of different origin. This restricted expression suggests FMNL1 to be an attractive target for novel immunotherapies in malignant and inflammatory diseases (2, 3). However, function and regulation of FMNL1 are less well characterized. Previous work has shown involvement of FMNL1 in the reorientation of the microtubuleorganizing center toward the immunological synapse and cytotoxicity of T cells (4). The murine homolog FRL, which has 85% homology to the human counterpart, has been additionally shown to be involved in cell adhesion and motility of macrophages as well as Fc␥ receptor-mediated phagocytosis (5, 6). To date, it is not clear how these different membrane-associated processes are regulated.Formins are defined by a unique and highly conserved C-terminal formin homology (FH) 2 domain that mediates the effects on actin (7-11). The FH2 domain is proceeded by a proline-rich FH1 domain that binds with low micromolar affinity to profilin (12, 13). In a conserved subfamily of formins known as diaphanous-related formins (DRFs), the FH1 and FH2 domains are flanked by an array of regulatory domains at the N terminus and by a single C-terminal diaphanous autoregulatory domain (DAD) (14). The large N-terminal regulatory region includes a binding domain for small G proteins like RhoGTPase followed by an adjacent diaphanous-inhibitory domain (DID) and a dimerization domain (13,(15)(16)(17). The DAD, which comprised only a small stretch of amino acid residues, binds to the DID. Interaction of DA...

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

“…Whereas mDia1 and mDia2 employ clusters of positively charged residues for anchoring of the formins via electrostatic interactions with acidic phospholipids to the plasma membrane (45,46), FMNL1 inserts into the membrane after being myristoylated at its N terminus (47). Next, we therefore sought to determine whether the GBD of ForC also plays a critical role for subcellular localization.…”

Protein N-Myristoylation Is Required for Cellular Morphological Changes Induced by Two Formin Family Proteins, FMNL2 and FMNL3

“…32) As for the protein N-myristoylation occurs in formin family proteins, it was found recently that FMNL1, a newly identified splice variant of FMNL1, is Nmyristoylated, and that N-myristoylation is required for plasma membrane localization and for the membrane bleb formation induced by FMNL1. 37) However, two previously identified isoforms of FMNL1, FMNL1 and FMNL1, neither localized to the plasma membrane nor induced cellular morphological changes when expressed in mammalian cells. In FMNL1 and FMNL1, however, similar membrane localization and bleb formation was observed for a mutant lacking the DAD domain (FMNL1ÁDAD), indicating that deregulation of autoinhibition is effective in FMNL1.…”

Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex

“…FMNL1 expression has been observed in T cells, B cells, malignant B cells from patients with chronic lymphocytic leukemia (CLL) and macrophages (Gomez et al, 2007;Han et al, 2009;Mersich et al, 2010;Schuster et al, 2007). However, northern blot analysis has shown FMNL1 expression in nonhematopoietic malignant cells (Krackhardt et al, 2002).…”

“…These alternatively spliced isoforms of FMNL1 differ in their C-termini. Interestingly, the FMNL1c isoform was reported to localize to the plasma membrane (PM), to regulate membrane blebbing and to be constitutively active (Han et al, 2009). FMNL1c was also found to regulate podosome dynamics and adhesion in macrophages (Mersich et al, 2010).…”