A Novel Integrin-activated Pathway Forms PKB/Akt- stimulatory Phosphatidylinositol 3,4-Bisphosphate via Phosphatidylinositol 3-Phosphate in Platelets

Abstract

The aggregation of human platelets is an important physiological hemostatic event contingent upon receptordependent activation of the surface integrin ␣ IIb ␤ 3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). It has been assumed that PtdIns(3,4)P 2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P 3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P 2 is formed after directly activating ␣ IIb ␤ 3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P 3 , but to transient formation of PtdIns3P and generation of PtdIns(3,4)P 2 , the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P 2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P 3 . Integrin-activated generation of the second messenger PtdIns(3,4)P 2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.Human platelets have provided a model system for a variety of signal transduction events, including integrin-based signaling. Platelets can be activated by agents that include agonists for the thrombin receptor (THR-R), 1 leading to a change in integrin ␣ IIb ␤ 3 conformation to one that binds plasma fibrinogen (FIB) and results in aggregation. The change in integrin is dependent partially upon the activation of an 85 K D subunit-containing form of PI3K (1, 2), which acts in vivo on PtdIns(4,5)P 2 and rapidly generates PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , but not PtdIns3P (3, 4). Late (post-aggregation) accumulations of PtdIns(3,4)P 2 , but not the levels of PtdIns(3,4,5)P 3 (5), have been found to be regulated by extracellular Ca 2ϩ and binding of FIB to ␣ IIb ␤ 3 (5, 6). Other work has shown that THR-R-dependent accumulation of PtdIns(3,4)P 2 can be impaired by calpeptin, an inhibitor of the Ca 2ϩ -dependent protease calpain, which is activated under these conditions (7-9). Norris et al. (9) have suggested that calpain hydrolytically inactivates PtdIns(3,4)P 2 4-phosphatase, thereby elevating PtdIns(3,4)P 2 . The rise in PtdIns(3,4)P 2 that follows THR-R stimulation has been correlated kinetically with the regulation of the serine-threonine kinase PKB/Akt (10), although a role for the earlier elevation in PtdIns(3,4,5)P 3 levels could not be discounted by these studies. Indeed, both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are potent stimuli for PDK1, which phosphorylates PKB/Akt and thereby activates it (11). Another report has a...

Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities

“…However, the finding that agonists, such as hydrogen peroxide [30], and crosslinking of platelet-integrin receptors [31] elevate PtdIns(3,4)P # , without increasing PtdIns(3,4,5)P $ , suggest that PtdIns(3,4)P # may be able to regulate physiological processes distinct from those controlled by PtdIns(3,4,5)P $ . TAPP1 and TAPP2 ( Figure 3) are the first proteins to be identified that interact with PtdIns(3,4)P # specifically, and may therefore be key mediators of cellular responses that are regulated specifically by this second messenger.…”

Coordinated Signaling through Both G12/13 and Gi Pathways Is Sufficient to Activate GPIIb/IIIa in Human Platelets

“…Major signaling molecules lying downstream of G protein activation include PKC (4), MEKK1 (41), PI 3-kinase (25,26), and p160 ROCK (23,24), among many others (3). We measured the effects of selective inhibitors for these molecules on platelet aggregation stimulated by combined G 12/13 and G i signaling.…”

“…The ␣-subunit of the heterotrimeric G protein G i pathway inhibits the activity of adenylyl cyclase while the ␤␥-subunit activates PI 3-kinase (25). Together, these pathways lead to the activation of numerous kinases including protein kinase B (PKB/Akt) (26), PKC (4), Map kinase kinase (MEKK1) (27), Src family tyrosine kinases (28), among many others.…”

The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane

“…4,4,5)P 3 ] is a transient plasma membrane (PM) signal, rapidly hydrolyzed to phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P 2 ] by the inositol polyphosphate 5-phosphatases (5-phosphatases) (Stephens et al, 1993;Mitchell et al, 2002). PtdIns(3,4)P 2 is a more sustained signal lasting for up to 60 min, stimulated by B-cell activation, oxidative stress, or irreversible platelet aggregation (Banfic et al, 1998;Van der Kaay et al, 1999;Marshall et al, 2002).…”