Phosphatidylinositol 3-Kinase and Ca2+ Influx Dependence for Ligand-stimulated Internalization of the c-Kit Receptor

Abstract

c-Kit is a receptor tyrosine kinase and a member of the subfamily that includes the PDGF, 1 CSF-1, and flt-3/flk-2 receptors. Together with its ligand steel factor (SLF), c-Kit is a key controlling receptor for a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells. c-Kit is the gene product of the W locus in mice (1), and its ligand SLF is the product of the sl locus (2, 3). Mutations in either locus severely affect the growth and survival of stem cells of these tissues.As with other receptor tyrosine kinases, stimulation of the c-Kit receptor with SLF results in the recruitment and tyrosine phosphorylation of SH2-containing second messenger-generating enzymes (4) such as phospholipase C-␥ and PI3-kinase (5, 6). The phosphorylated lipid products of these enzymes stimulate a variety of intracellular processes including Ca 2ϩ mobilization and actin reorganization (7-9). Coincident with second messenger generation, the process of receptor internalization is also initiated. Within minutes following ligand binding, receptors cluster in dimers or oligomers and internalize by endocytosis, likely through clathrin-coated pits (10 -12). Eventually, clathrin coats are removed, and the remaining vesicles fuse with endosomes, late endosomes, and ultimately lysosomes, resulting in receptor degradation.Numerous deletion and mutagenesis studies have been carried out to map regions of receptor tyrosine kinases required for ligand-stimulated internalization (13-22). Tyrosine kinase activity (23, 24), autophosphorylation sites (17, 18), and interactions with second messenger-generating enzymes have been implicated. In particular, recruitment and activation of PI3-kinase have been associated with ligand-stimulated internalization of the PDGF receptor (14). Receptors carrying mutations within the cytoplasmic domain that disrupt the PI3-kinase-binding site are impaired in the later stages of endocytosis (25). However, conflicting results with a deletion mutant encompassing this binding site demonstrate no impairment in any stage of endocytosis (16).Ligand-stimulated endocytosis of c-Kit was investigated by Yee et al. (26). They reported that a c-Kit mutant with no kinase activity was impaired for ligand-stimulated internalization. Individual mutations converting tyrosines 719 and 821 to phenylalanines, however, failed to affect internalization, although Phe-821 was impaired for mitogenesis (27). Tyr-719 is located in the kinase insert region of the c-Kit catalytic domain. In its phosphorylated form, it forms part of a consensus binding site for the p85 subunit of PI3-kinase and has been shown to mediate this interaction in vitro and in vivo (6).We have also examined the role of PI3-kinase activation on ligand-stimulated internalization of c-Kit. We found that in the absence of PI3-kinase activation, the c-Kit receptor internalizes but remains localized near the inner aspect of the plasma membrane. However, when both PI3-kinase and Ca 2ϩ influx are inhibited, clathrin fails to co-immunoprec...

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

“…The soluble form of SCF rapidly activates c-Kit but the signalling activity quickly decays owing to receptor internalisation. The membrane bound form gives a more persistent signalling activity, possibly owing to increased stability of the receptor-ligand complex and reduced internalisation (Miyazawa et al, 1995;Gommerman et al, 1997). In vivo, only the membrane bound form supports survival of long-term haematopoietic progenitors (Gommerman et al, 2000;Driessen et al, 2003).…”

Protein Kinase C-ζ and Protein Kinase B Regulate Distinct Steps of Insulin Endocytosis and Intracellular Sorting

“…The role of PI3K in the endocytosis of other tyrosine kinase receptors has been shown previously (17)(18)(19), although the mechanisms remain unclear. Overexpression and antisense studies have now defined a specific role for PKC in insulin internalization.…”

“…PI3K is necessary for the internalization of several growth factor receptors (17)(18)(19). PI3K and its downstream effectors have been implicated in the trafficking of endocytic and exocytic vesicles in yeast and mammalian cells (20,21).…”

Bortezomib interferes with C-KIT processing and transforms the t(8;21)-generated fusion proteins into tumor-suppressing fragments in leukemia cells

“…C-KIT internalization is mediated by clathrin (3,4,23). We evaluated whether clathrin plays a role in BOR-induced C-KIT internalization using DY, a potent inhibitor of dynamin GTPase that is essential for clathrin-dependent coated vesicle formation (21).…”

“…On binding to its ligand, the stem cell factor (SCF), C-KIT rapidly undergoes dimerization, autophosphorylation (2), and clathrin-mediated internalization (3,4). Through its downstream signal molecules, including PI3K, Rac-serine/threonine-protein kinase (AKT), ERK, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC), JAK/ STAT, and Rat sarcoma (Ras)/Rapidly Accelerated Fibrosarcoma (Raf)/MAPK cascade (5), C-KIT confers survival/proliferative signals to hematopoietic stem cells, mast cells, germ cells, melanocytes, and interstitial cells of Cajal (6).…”