When Swiss 3T3 cells are treated with Insulin‐like Growth Factor I, a rapid decrease in the mass of polyphosphoinositol lipids (phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate) occurs within the nuclei, with a concomitant increase in nuclear diacylglycerol and translocation of protein kinase C to the nuclear region. This is in contrast to the effects of the regulatory peptide, bombesin, which causes similar inositol lipid changes in the plasma membrane, has no effect on nuclear inositide levels and causes a translocation of protein kinase C to post‐nuclear membranes. These results suggest the existence of a discrete nuclear polyphosphoinositide signalling system entirely distinct from the well‐known plasma membrane‐located system, which is under regulatory control by cell surface‐located receptors.

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...

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We have observed that aggregation of human platelets, caused by activation of integrin ␣ IIb ␤ 3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of phosphatidylinositol 3,4-bisphosphate, thereby activating protein kinase B/Akt. Such synthesis depends upon both the generation of phosphatidylinositol 3-phosphate (PtdIns3P), which is sensitive to wortmannin (IC 50 7 nM) and calpain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report that a recently characterized C2 domain-containing phosphoinositide 3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic cell line (CHRF-288) derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P observed in platelets. HsC2-PI3K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC 50 6 -10 nM), requires Mg 2؉ , and shows strong preference for PtdIns over PtdIns4P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is activated severalfold when platelets aggregate in an ␣ IIb ␤ 3 -dependent manner or when platelet or CHRF-288 lysates are incubated with Ca 2؉ . Activation is prevented by calpain inhibitors. CHRF-288, which cannot undergo activation of ␣ IIb ␤ 3 and thereby aggregate in response to platelet agonists, do not generate PtdIns3P or activate HsC2-PI3K under conditions that stimulate other phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector for integrin-dependent signaling.

Highly purified nuclei were prepared from livers and kidneys of rats undergoing compensatory hepatic or renal growth, the former being predominantly by cellular proliferation, and the latter mostly by cellular enlargement. In liver, an increase in nuclear diacylglycerol (DAG) concentration occurred between 16 and 30 h, peaking at around 20 h. At the peak of nuclear DAG production a specific translocation of protein kinase C to the nucleus could be detected; no such changes occurred in kidney. There was no detectable change in whole-cell DAG levels in liver, and the increase in DAG was only measurable in nuclei freed of their nuclear membrane. Overall, these results suggest that there is a stimulation of intranuclear DAG production, possibly through the activation of an inositide cycle [Divecha, Banfić and Irvine (1991) EMBO J. 10, 3207-3214] during cell proliferation in vivo.