Lee A W, States D J
Departments of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 2000 Sep;20(18):6779-98. doi: 10.1128/MCB.20.18.6779-6798.2000.
Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.
集落刺激因子1（CSF-1）支持骨髓来源的单核细胞系细胞的增殖、存活和分化。在表达CSF-1受体（CSF-1R）的髓系祖细胞32D细胞系中，CSF-1对细胞外信号调节激酶（ERK）途径的激活既依赖于Ras又依赖于磷脂酰肌醇3激酶（PI3激酶）。PI3激酶抑制并不影响导致Ras激活的事件。以PI3激酶效应器Akt的活性作为读数，用显性负性和致癌性Ras进行的研究未能将PI3激酶置于Ras的下游。因此，PI3激酶似乎与Ras平行发挥作用。PI3激酶抑制剂增强了CSF-1刺激的A-Raf和c-Raf-1活性，显性负性A-Raf而非显性负性c-Raf-1降低了CSF-1引发的ERK激活，这表明A-Raf介导了从Ras到MEK/ERK的部分刺激信号，与PI3激酶平行发挥作用。出乎意料的是，缺乏PI3激酶结合位点的CSF-1R（DeltaKI）仍能够以PI3激酶依赖的方式激活MEK/ERK。为了确定Src家族激酶（SFKs）是否参与其中，我们证明CSF-1在表达野生型（WT）或DeltaKI受体的细胞中激活了Fyn和Lyn。此外，在表达DeltaKI的细胞中，CSF-1诱导的Akt活性依赖于SFK，因为SFK活性的药理学或遗传学抑制可阻止Akt激活。对接蛋白Gab2可能将SFK与PI3激酶联系起来。CSF-1在表达WT或DeltaKI受体的细胞中诱导Gab2酪氨酸磷酸化并与PI3激酶结合。然而，只有在DeltaKI细胞中，这些事件才会被PP1阻止。因此，在髓系祖细胞中，CSF-1可通过至少两种机制激活PI3激酶/Akt途径，一种涉及直接受体结合，另一种涉及SFKs。
