Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages

Abstract

Loss-of-function mutations in the murine dominant white spotting ͞c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase͞mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.T he Kit receptor tyrosine kinase (RTK) is centrally involved in the development of multiple cell lineages, including hematopoietic and germ cells, melanocytes, and the interstitial cells of Cajal (ICC) (1-4). Insights into the roles of this receptor and its cognate ligand, stem cell factor (SCF), in these developmental processes have been greatly facilitated by the large series of naturally occurring mutations in the murine genes that encode these molecules, the dominant white spotting (W) and steel (Sl) loci, respectively. Thus, mice with loss-of-functions in either the W or Sl loci are anemic and exhibit white spotting, sterility, and a concomitant loss of the ICC and intestinal pacemaker activity.Ligand binding to the Kit RTK induces receptor dimerization and autophosphorylation of specific tyrosine residues (5, 6). These phosphorylation events create docking sites for specific Src homology 2 (SH2) domain-containing proteins, which in turn control various intracellular signaling pathways (7). Recruitment of particular targets is mediated by the ability of their SH2 domains to recognize specific phosphotyrosine (pTyr)-containing motifs on the activated receptor (8). Numerous signaling molecules have been identified as binding partners for specific pTyr residues on activated Kit, including the p85 subunit of phosphatidylinositol 3Ј kinase (by means of tyrosine 719), phospholipase C␥ (by means of tyrosine 728), and the Grb2 and Grb7 adapter proteins (by means of tyrosine residues 702 and 934) (6). Additionally, signaling molecules, including Src family kinases and the protein tyrosine phosphatases Shp-1 and Shp-2, have been shown to ...

The Scaffolding Adapter Gab2, via Shp-2, Regulates Kit-evoked Mast Cell Proliferation by Activating the Rac/JNK Pathway

“…8). Two recent papers showed that there are Ͼ95% deceases of mast cells in the peritoneal cavity (6,43) and stomach (43) of Kit Y567F knock-in mice. The loss of mast cells in these two tissues in the Kit Y567F/Y567F mice resembles the phenotypes that were observed in Gab2 Ϫ/Ϫ mice (32).…”

“…However, analysis of mast cells in hypodermis (Fig. 7B), stomach (32), and peritoneal cavity (6,30,32,43) (data not shown) suggest that the Kit Tyr 567 -Gab2 activated pathway is more important than the Kit Tyr 719 pathway in mast cell development in these specific tissues. One possibility is that the signal from Kit Tyr 719 might be transient or weak.…”

Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells

“…5,7,8 Mutational and "knock-in" studies on Kit Y567/Y569 have revealed their important role in cell development, proliferation, survival, and migration. 6,[9][10][11] However, the biologic significance of these interactions is still poorly understood.…”

SH2 Domain-Containing Phosphatase 2 Is a Critical Regulator of Connective Tissue Mast Cell Survival and Homeostasis in Mice

“…Further studies using knock-in mutations of individual KIT tyrosine residues identified key signaling pathways required for mast cell development (1,24,25,45,48). Phosphorylation of KIT Y719 allows recruitment of the p85␣ regulatory subunit of class I A phosphatidylinositol 3-kinase (PI3K) (44).…”