Andrius Kazlauskas

Growth-factor-dependent mitogenesis requires two distinct phases of signalling

Prolonged and continuous exposure to growth factors is required to commit cells to the cell cycle. Here we show that the prolonged requirement for growth factor can be replaced with two short pulses of mitogen. The first pulse of growth factor moves the cell through the initial segment of the G0 to S interval. This initial pulse also makes cells responsive to a second pulse of growth factor, which engages components of the cell-cycle machinery necessary for progression into S phase. We also show that activation of MAP kinase kinase (MEK) and induction of the transcription factor c-Myc are sufficient to drive the first, but not the second, phase of signalling. Furthermore, synthetic phosphatidylinositol-3-OH kinase (PI(3)K) lipid products are sufficient to drive the second phase of signalling, but not the first. These findings suggest that there is a common signalling cascade by which mitogens drive arrested cells into the cell cycle, and that this cascade involves the temporally coordinated input of MEK, c-Myc and PI(3)K.

Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes.

Platelet-derived growth factor (PDGF) acts as a potent mitogen, chemoattractant and survival factor for mesenchymal cells. In addition to its importance in mammalian development, PDGF plays a critical role in physiological repair mechanisms and in the pathogenesis of various proliferative diseases. The biological effects of PDGF are initiated via two related receptor tyrosine kinases, termed alpha and betaPDGF receptors. Recent observations provide increasing evidence for distinct roles of the two PDGF receptor subtypes in both embryogenesis and disease formation. Moreover, characterization of the signal relay mechanisms indicates, that the alpha and betaPDGF receptors are not identical in their ability to bind intracellular effector molecules. Furthermore, the two PDGF receptors initiate overlapping, yet distinct signal transduction pathways. These differences may account for some of the variabilities in biological responses resulting from activation of these two receptors.

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each others binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.

Receptor tyrosine kinases and their targets.

One of the ways in which higher eukaryotes receive messages from the environment is via cell surface receptor tyrosine kinases. These are transmembrane proteins with an extracellular binding domain that specifies the growth factor with which it will interact, and an intracellular domain that encodes the tyrosine kinase. The mechanism by which receptor tyrosine kinases direct intracellular signal relay appears to involve receptor autophosphorylation that permits the stable binding of SH2 domain containing signal transduction enzymes. Some of the more recent advances are summarized in this review.

Vascular Endothelial Growth Factor and Hepatocyte Growth Factor Levels Are Differentially Elevated in Patients with Advanced Retinopathy of Prematurity

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.

PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1

BACKGROUND Platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis within minutes. These events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully account for later responses, such as cell-cycle progression. RESULTS We assessed when PI 3-kinase products accumulate in PDGF-stimulated cells. In addition to the previously identified early accumulation of products, we detected a second, prolonged wave of accumulation 3-7 hours after stimulation. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis, we first developed an assay in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a PDGF-receptor mutant. The lipids rescued DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors failed to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1 phase, prevented PDGF-dependent cell-cycle progression. CONCLUSIONS PDGF induces distinct waves of PI 3-kinase activity. The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use PI 3-kinase to mediate distinct cellular responses seems to be by regulating when its products accumulate.

A new link between the c-Abl tyrosine kinase and phosphoinositide signalling through PLC-γ1

The c-Abl tyrosine (Tyr) kinase is activated after platelet-derived-growth factor receptor (PDGFR) stimulation in a manner that is partially dependent on Src kinase activity. However, the activity of Src kinases alone is not sufficient for activation of c-Abl by PDGFR. Here we show that functional phospholipase C-γ1 (PLC-γ1) is required for c-Abl activation by PDGFR. Decreasing cellular levels of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by PLC-γ1-mediated hydrolysis or dephosphorylation by an inositol polyphosphate 5-phosphatase (Inp54) results in increased Abl kinase activity. c-Abl functions downstream of PLC-γ1, as expression of kinase-inactive c-Abl blocks PLC-γ1-induced chemotaxis towards PDGF-BB. PLC-γ1 and c-Abl form a complex in cells that is enhanced by PDGF stimulation. After activation, c-Abl phosphorylates PLC-γ1 and negatively modulates its function in vivo. These findings uncover a newly discovered functional interdependence between non-receptor Tyr kinase and lipid signalling pathways.

Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

Growth factor-dependent signaling and cell cycle progression

There are three central ideas contained within this review. Firstly, growth factor‐stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.

Axl is essential for VEGF-A-dependent activation of PI3K/Akt

Herein, we report that vascular endothelial growth factor A (VEGF‐A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF‐A‐dependent activation of VEGF receptor‐2 (VEGFR‐2), and subsequent TSAd‐mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand‐independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF‐A‐mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF‐A‐ but not bFGF‐dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF‐A‐induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF‐A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF‐A‐driven processes.