The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.

SUMMARY Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or β (PI5P4Kα and β) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and β in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K β and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A−/−, PIP4K2B+/−, and TP53−/− mice were viable and had a dramatic reduction in tumor formation compared to TP53−/− littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P 2 , a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P 2 in the DMS occurs in proximity to PI-5-P-4-kinase ␣ (PIP4K␣), and short hairpin (sh) RNAmediated loss of PIP4K␣ impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P 2 is a marker and possibly essential component of internal membranes. PI-4,5-P 2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P 2 in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P 2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis. IntroductionMammals synthesize blood platelets as functional cell particles within a precursor cell, the megakaryocyte (MK). Terminally differentiated MKs acquire a significantly polyploid DNA content and enlarge to 50 to 100 m in diameter before releasing their platelet load. In the release phase, the MK cytoplasm converts into long branched protrusions, and disc-shaped platelets are assembled de novo within these proplatelet extensions. 1 Microtubule bundles form the core of each proplatelet, and their distal tips organize into the repetitively coiled structure of the platelet marginal band. [1][2][3] The actin cytoskeleton also participates actively in thrombopoiesis, judged mainly by the effects of inhibitors of actin polymerization on proplatelet morphology. 1,[4][5][6] Although these studies reveal cytoskeletal aspects of thrombopoiesis, little is known about the corresponding regulation of nascent platelet membranes or how membrane and cytoskeletal morphogenesis are coordinated.The conversion of a single MK into thousands of platelets is accompanied by a large increase in total surface area. MK ultrastructure reveals an abundant pool of cytoplasmic membranes that constitute the demarcation membrane system (DMS). By virtue of its origin in tubular invaginations of the plasma membrane, the DMS maintains continuity with the extracellular space, 7,8 and whole-cell patch-clamp recordings reveal the DMS to be a single electrophysiologic entity. 9 DMS functions, however, remain controversial, and its name recalls early theories on platelet biogenesis. Prior to proplatelet-based models of thrombopoiesis, nascent platele...