Differential Role of Class 1A PI 3-Kinase Regulatory Subunits in Mast Cell Growth, Maturation and Survival.

Abstract 77

Stem cell factor (SCF) mediated c-Kit receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from c-Kit are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI 3-kinase including p85α, p85β, p50α, and p55α. While it is known that PI 3-kinase plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including maturation, growth, and survival are not known. Using mice deficient in the expression of p85α or p85β or combination of both p85α/p55α/p50α as well as all four subunits we have examined the role of these subunits in mast cell functions. We show that loss of p85α subunit alone results in impaired bone marrow derived mast cell (BMMC) maturation, growth, and survival compared to wild-type (WT) controls, in spite of the continuous expression of p85β, p55α, and p50α subunits in these cells. Restoring the expression of p85α in p85α deficient mast cells restores the maturation and growth defects. To assess the contribution of p50α and p55α subunits, we generated mice using the Cre lox system that were deficient in the expression of all three subunits (i.e. p85α/p55α/p50α). Deficiency of p85α/p55α/p50α subunits in bone marrow cells completely blocked mast cell maturation and growth, suggesting an essential role for the smaller subunits p50 and p55 in addition to the full length form of p85. Curiously, over-expression of p50α in p85α deficient BMMCs only marginally rescued mast cell maturation and growth, suggesting that the full length form of p85α functions with specificity in regulating mast cell functions. Since the major difference between the shorter isoforms and the full length form of p85α is the absence of the amino terminal SH3 and BH domains, we generated two mutants of p85α lacking either the SH3 or the BH domain and expressed them in p85α−/− BMMCs. While both these mutants completely restored the maturation defect associated with p85α deficiency and showed normal binding to the c-Kit receptor upon SCF stimulation as well as to the p110 catalytic subunits; none of these mutants completely rescued SCF induced proliferation (50% and 70% respectively, n=3, p<0.004). Biochemically, lack of SCF induced growth rescue in p85α−/− BMMCs expressing p85αΔSH3 and p85αΔBH mutants was associated with a lack of rescue in the activation of Akt and Erk, but complete rescue in the activation of JNK (n=3). Consistently, while transplantation of p85α deficient bone marrow cells transduced with p85α into mast cell deficient Wsh mice resulted in complete restoration of gastrointestinal mast cells as well as mast cells in the stomach and spleen, p85αΔSH3 and p85αΔBH mutants restored mast cells only partially. These results indicate that other domains (SH3 and BH) of p85α are required for mast cell growth. In contrast to p85α, deficiency of p85β alone resulted in increased BMMC maturation, growth and survival compared to controls (1.2 fold, n=3, p<0.003). Consistently, over-expression of p85β in WT bone marrow cells resulted in a profound reduction in the maturation of mast cells as well as proliferation. We studied whether reduced maturation and proliferation due to the loss or over-expression of p85β was a result of altered c-Kit receptor internalization and degradation. Our results revealed significantly more c-Kit receptor internalization and degradation in p85β expressing cells compared to p85α expressing cells (2 fold, n=5, p<0.001). Since Cbl family of ubiquitin ligases are involved in the down-regulation of tyrosine kinase receptors, we analyzed whether c-Cbl is involved in p85β mediated c-Kit receptor internalization and degradation. Phosphorylation of c-Cbl and ubiquitination of c-Kit receptor was more in p85β expressing cells compared to p85 expressing cells (n=3). In conclusion, while the current dogma in the field of PI3Kinase signaling suggests that all regulatory subunits of PI3Kinase function in a similar manner; we provide genetic and biochemical evidence to suggest that p85 regulatory subunits differentially regulate growth and maturation of mast cells.