Hiregowdara D, Avraham H, Fu Y, London R, Avraham S
Division of Experimental Medicine, Beth Israel Deaconess Medical Center (West Campus), Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1997 Apr 18;272(16):10804-10. doi: 10.1074/jbc.272.16.10804.
We have characterized signaling pathways involving the related adhesion focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-beta) in CMK human megakaryocytic cells. Stem cell factor, which potentiates the growth of megakaryocytes and their progenitors, and phorbol myristate acetate, which causes differentiation of megakaryocytic cell lines, induced the tyrosine phosphorylation of RAFTK but not of focal adhesion kinase. Stimulation of CMK cells with stem cell factor resulted in an increase in the autophosphorylation and kinase activity of RAFTK. Phosphorylation of RAFTK under these conditions was mediated by a protein kinase C-dependent pathway. Cytochalasin D, which disrupts the cytoskeleton, abolished the phosphorylation of RAFTK upon phorbol myristate acetate and stem cell factor stimulation, indicating that RAFTK association with the actin cytoskeleton appears to be critical for its phosphorylation. In addition, we observed an association of RAFTK with paxillin, a 68-kDa cytoskeleton protein. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly through the C-terminal proline-rich domain. Transient overexpression of a dominant-negative mutant of RAFTK inhibited significantly the tyrosine phosphorylation of paxillin upon phorbol myristate acetate stimulation. These observations indicate that RAFTK might play an important role in the phosphorylation of signaling pathways within the focal adhesions and that RAFTK participates in signaling events that link signals from the cell surface to the cytoskeleton. Furthermore, this study suggests that RAFTK might be involved in megakaryocyte proliferation and differentiation.
我们已经在CMK人巨核细胞中对涉及相关黏附灶性酪氨酸激酶（RAFTK，也称为PYK2或CAK-β）的信号通路进行了表征。干细胞因子可增强巨核细胞及其祖细胞的生长，佛波酯肉豆蔻酸酯可导致巨核细胞系分化，二者均可诱导RAFTK的酪氨酸磷酸化，但不诱导黏附斑激酶的酪氨酸磷酸化。用干细胞因子刺激CMK细胞会导致RAFTK的自身磷酸化和激酶活性增加。在这些条件下，RAFTK的磷酸化由蛋白激酶C依赖性途径介导。细胞松弛素D可破坏细胞骨架，它消除了佛波酯肉豆蔻酸酯和干细胞因子刺激后RAFTK的磷酸化，这表明RAFTK与肌动蛋白细胞骨架的结合似乎对其磷酸化至关重要。此外，我们观察到RAFTK与桩蛋白（一种68 kDa的细胞骨架蛋白）存在关联。通过体外结合试验表明，RAFTK和桩蛋白可通过富含脯氨酸的C末端结构域直接结合。RAFTK显性负性突变体的瞬时过表达显著抑制了佛波酯肉豆蔻酸酯刺激后桩蛋白的酪氨酸磷酸化。这些观察结果表明，RAFTK可能在黏附斑内信号通路的磷酸化中发挥重要作用，并且RAFTK参与了将细胞表面信号与细胞骨架联系起来的信号转导事件。此外，这项研究表明RAFTK可能参与巨核细胞的增殖和分化。
