Molecular and cellular regulation

The general mechanisms of ligand-induced activation of Kit/SCF-R and its downstream signaling have been fairly well characterized, and very similar principles seem to govern ligand-induced signaling by all subclass III RPTKs. In outline, binding of the bivalent ligand, SCF, induces dimerization of Kit/SCF-R leading to activation of its tyrosine kinase domain and subsequent autophosphorylation on specific tyrosine residues. The phosphorylated tyrosine residues and three to five amino acid residues immediately N- or C-terminal to each of these, in turn, create specific binding sites for intracellular signaling molecules leading to their recruitment from cytosolic compartments to the membrane, where the activated RPTK is located. The binding of signaling molecules to the phosphorylated tyrosine residues occur through conserved protein.protein interaction domains, such as Src homology 2 (SH2) and protein tyrosine binding (PTB) domains. The SH2 and PTB domains have a defined, conserved primary sequence of 100 and 110 amino acids, respectively, and they exist as independently folded domains in a variety of intracellular proteins including proteins possessing a catalytic domain, pure adaptor molecules, structural proteins, and translocated transcription factors. The conformational changes induced in the signaling molecules upon binding to an RPTK can unmask other protein.protein interaction domains allowing for interaction, recruitment, and activation of yet other intracellular signaling molecules. Several other protein.protein interaction domains that have been identified include prolinerich- binding SH3 and WW domains, phospholipidbinding PH (pleckstrin homology) domains, and phosphoserine/phosphothreonine-binding 14-3-3 (.14-3-3 Proteins) and FHA (Forkhead-associated) domains. The specificity of signaling from different RPTKs is to a large extent determined by the primary sequence surrounding the autophosphorylated tyrosine residues in the receptors. This enables the activated RPTK to ЃgselectЃh a specific subset of signaling molecules within the cell, which, in turn specifically interact with, and/or activate, other signaling molecules, and a network of activated catalytic proteins and of multiprotein signaling complexes result. Examples of Kit/SCF-R-induced signaling molecules and pathways of importance for cell growth control include the Ras- Raf-ERK and Src-activated pathways, which are mainly involved in cell proliferation and the phosphatidylinositol 30-kinase (.PI3-Kinase) and JAK/STAT (.Signal Transducers and Activators of Transcription in Oncogenesis) signaling pathways involved in cell proliferation and cell survival (Fig. 1). Several signaling components within each of these pathways are able to interact with and/or modify the activity of each other, allowing for so-called .signal transduction cross-talk. This is important for finetuning and modulation of signaling. It is becoming increasingly clear that such Kit/SCF-R-initiated signaling pathways eventually impinge on and regulate the cell cycle machinery including the .p16-cyclin D-.RB pathway, as well as the DNA damage response pathways including p19ARF and p53. It is perhaps exclusively through these latter effects that the signaling pathways ultimately regulate proliferation and/or anti-apoptosis, respectively. For some recent reviews on RPTK-initiated signal transduction and mitogenic/survival signaling pathways [2, 3].

Once the appropriate Kit/SCF-R-induced signaling pathways have been activated, it is crucial that further Kit/SCF-R signaling is downregulated and inactivated to achieve a proper cell biological response. Several mechanisms prevail to achieve this, including negative feedback mechanisms, ligand-induced receptor internalization and degradation, and coordinated activation or organization of negative intracellular regulators including phosphatases and scaffolding proteins. In the case of Kit/SCF-R, SCF stimulation causes activation of.PKC, which acts in a negative feedback loop to inhibit further Kit/SCF-R tyrosine kinase activity by directly phosphorylating two serine residues in the receptor (Fig. 1). SCF stimulation also induces Kit/ SCF-R internalization with a T. < 0.5 h, and it has been shown that the internalized receptors undergo regulated degradation, in part through .ubiquitinmediated proteolysis. The ligand-induced Kit/SCF-R downregulation is likely to play an important regulatory physiological role in development, since SCF-expressing stromal cells interact with Kit/SCFR- expressing parenchymal cells in gonads, bone marrow, and cerebellum during development. In these tissues SCF exists in a mainly transmembrane form, which does not induce Kit downregulation to the same extent as the soluble form of SCF, thus allowing for more sustained Kit signaling. There is some evidence that there are quantitative and qualitative differences in the activated signaling molecules induced by the two forms of SCF. The transmembrane form of SCF is important for stem cell self-renewal and survival, while the soluble form is important for chemotaxis and cell proliferation. As part of a long-term mechanism to ensure proper Kit/SCF-R signaling, the Kit expression level is regulated in a cell- and tissuespecific manner at the transcriptional level as well. The c-kit promoter contains Sp1, SCL/Tal1, and AP-2-binding sites and its expression is tightly and positively regulated by each of these transcription factors.

Perturbation of the normal regulatory mechanisms governing Kit/SCF-R-induced signal transduction has serious clinical consequences. Accordingly, both in humans and mice, naturally occurring loss-offunction mutations and gain-of-function mutations in Kit/SCF-R have been reported that result in developmental defects or malignancies.


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