Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

Abstract

Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production.

Symposium review: Intravital imaging of the lactating mammary gland in live mice reveals novel aspects of milk-lipid secretion

“…Immunocytochemical studies have shown an association of actin with budding lipid droplets (Franke et al, 1981), and an actin meshwork associated with the apical surface and secretory apparatus was revealed by decoration with heavy meromyosin (Amato and Loizzi, 1981). Furthermore, lipid secretion was inhibited in mammary cells that were null for actin-filament associated protein, an adaptor protein that functions through cSrc in the reorganization of the actin cytoskeleton (Cunnick et al, 2015). In partial agreement with these studies, phalloidin staining of fixed tissue from the GFP membr mouse and intravital imaging of GFP LifeAct and GFP myoIIa mice revealed an extensive filamentous network of actin and myosin IIa in associa-tion with lipid droplets at the apical surface ( Figure 9A, B, and C).…”

Long non‐coding RNA AFAP1‐AS1 is upregulated in a subset of multiple sclerosis patients

“…8 Another area of research in the pathogenesis of MS involves the weakened function of the bloodbrain barrier (BBB). 11 Notably, a naturally occurring antisense has been recognized to be transcribed from the antisense strand of AFAP1. 9 Src family kinases participate in numerous signaling pathways, and transmit signals to the cytoskeleton, membrane and nuclear targets.…”

“…10 One of the Src binding partners is encoded by the actin filament associated protein 1 (AFAP1) gene. 11 Notably, a naturally occurring antisense has been recognized to be transcribed from the antisense strand of AFAP1. This lncRNA has an established function in the pathogenesis of diverse kinds of cancers.…”

AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts

“…AFAP1 is upregulated in certain cancers and AFAP1 expression is associated with higher grades of prostate cancer [ 38 ]. Using AFAP1 -/- null mice we were the first to demonstrate a novel physiological role for AFAP1 in lactation [ 39 ]. These studies demonstrated that AFAP1 is required for the spatial and temporal regulation of cSrc activity in the normal breast during lactation to establish copious milk production at parturition and, specifically, required for milk fat production [ 40 ].…”

“…Using AFAP1 -/- null mice we were the first to demonstrate a novel physiological role for AFAP1 in lactation [ 39 ]. These studies demonstrated that AFAP1 is required for the spatial and temporal regulation of cSrc activity in the normal breast during lactation to establish copious milk production at parturition and, specifically, required for milk fat production [ 40 ]. Although we are beginning to understand a physiological role of AFAP1 and its role in directing cSrc activity in the normal breast, potential roles for AFAP1 in other tissues and cells with abundant expression of AFAP1 have yet to be characterized.…”