Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. We demonstrate that distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases were associated with distinct subnuclear domains, identified as "nuclear speckles," which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures.

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Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly. Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms. Using peptide sequences from the erythroid 68-kDa PIP5KI, we have isolated cDNAs encoding PIP5KI␣ from human brain. Partial cDNAs obtained for a second isoform, PIP5KI␤, established that the human STM7 gene encoded a previously unrecognized PIP5KI. However, the peptide sequences demonstrated that erythroid PIP5KI corresponded to PIP5KI␣. Recombinant, bacterially expressed PIP5KI␣ possessed PIP5K activity and was immunoreactive with erythroid PIP5KI antibodies. By Northern analysis, PIP5KI␣ and PIP5KI␤ had wide tissue distributions, but their expression levels differed greatly. PIP5KIs had homology to the kinase domains of PIP5KII␣, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base. These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes. Furthermore, the limited homology between PIP5KIs and PIP5KII␣, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks. Phosphatidylinositol-4-phosphate 5-kinases (PIP5K)1 synthesize phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) by phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P). This conversion is pivotal to the phosphoinositide cycle as PtdIns(4,5)P 2 is hydrolyzed by phosphoinositide-specific phospholipase C to generate the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. These second messengers activate several protein kinase C isoforms and effect the release of stored intracellular calcium, respectively. PtdIns(4,5)P 2 is also phosphorylated by agonist-activated phosphatidylinositol 3-kinases, resulting in synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger whose targets are largely unknown, but may include protein kinase C isoforms (1-3). In addition, PtdIns(4,5)P 2 directly modulates the function of proteins, including phospholipase D (4) and many actin-binding proteins (5). It binds pleckstrin homology domains found in some signaling proteins (6, 7) and is required for Ca 2ϩ -regulated secretion of neurotransmitters (8). The diversity of PtdIns(4,5)P 2 actions suggests that PIP5Ks are crucial enzymes for many cellular functions (reviewed in Ref. 9).Multiple PIP5K isoforms have been purified and biochemically characterized (9 -13). The best characterized isoforms are the erythroid enzymes which have been categorized as type I or type II PIP5Ks based on their separation by phosphocellulose ion exchange (10 -12). These PIP5Ks differ in molecular mass and are biochemically and immunologically distinct. Two notable differences are that PIP5KI, but not PIP5KII, can be stimulated by pho...

ASAP1 (ADP ribosylation factor [ARF]-GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src . Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK. INTRODUCTIONAttachment of cells to the extracellular matrix (ECM) is primarily mediated by the integrin family receptors (Hynes, 1992). Engagement of heterodimeric integrin receptors leads to the clustering of integrins and recruitment of numerous proteins to form multi-protein complexes on the cytoplasmic face of the plasma membrane termed focal adhesions (Burridge et al., 1988). Focal adhesions serve to anchor actin cytoskeleton to the plasma membrane and to provide a linkage between the extracellular environment and the cytoplasm (Burridge and Chrzanowska-Wodnicka, 1996). The recruitment of cytoskeletal proteins and the assembly of focal adhesions are functionally important for a number of cellular processes, including cell migration, survival, and proliferation (Lauffenburger and Horwitz, 1996). In the case of migrating cells (or cells spreading on ECM proteins), there is a requirement for the coordinated reorganization of the actin cytoskeleton and the formation of new attachments with the substratum (Huttenlocher et al., 1995;Bretscher, 1996). This process is temporally and spatially controlled, consistent with integrins functioning as both cell adhesion receptors and as initiators of signaling cascades that convey signals from ECM to actin cytoskeleton (Bretscher, 1996).Because integrins are catalytically inactive, their signaling ability is dependent upon the recruitment and activation of Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-01-0018. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-01-0018.* Corresponding a...

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.Two distinct pathways have been characterized for agoniststimulated signal transduction involving phosphatidylinositol (PtdIns).1 One pathway entails activation of phosphatidylinositol-specific phospholipase C by extracellular agonists resulting in the hydrolysis of phosphoinositides to generate soluble inositol phosphates including inositol 1,4,5-trisphosphate and diacylglycerol (reviewed in Refs. 1 and 2). The other pathway involves receptor-mediated activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) to produce the second messengers, phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) (reviewed in Refs. 3 and 4).A pathway for the formation of D-3-phosphatidylinositols, proposed based on kinetic studies of intact human neutrophils, is through phosphorylation of the D-3 position of the myoinositol ring of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) by a PtdIns 4,5-P 2 3-kinase and subsequent dephosphorylation of PtdIns 3,4,5-P 3 to produce PtdIns 3,4-P 2 (5). This pathway has been supported by the existence of the extensively characterized PtdIns 3-kinase enzyme family, which can catalyze in vitro phosphorylation of phosphatidylinositol 4-phosphate (PtdIns 4-P) and PtdIns 4,5-P 2 . Evidence for a different pathway for the formation of D-3 phosphatidylinositols has been found in human platelets, NIH 3T3 cells, and plants in which phosphorylation of the D-3-position of PtdIns to form PtdIns 3-P is followed by phosphorylation of the D-4-position to give PtdIns 3,4-P 2 and then of the D-5-position to form PtdIns 3,4,5-P 3 (6 -

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP 2 from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKI␣ was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKI␣ in particle engulfment, we generated a double mutant (D309N/ R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KI␣ D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fc␥ receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP 2 and of F-actin in the phagosomal cup. These data suggest that PIPKI␣ activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKI␣ appears to contribute to the transient changes in PIP 2 levels that are associated with, and likely required for, the recruitment and regulation of actinmodulating proteins.

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Casein kinase I is a highly conserved family of serine/threonine protein kinases present in every organism tested from yeast to humans. To date, little is known about the function of the higher eukaryotic isoforms in this family. The CKI isoforms in Saccharomyces cerevisiae, however, have been genetically linked to the regulation of DNA repair, cell cycle progression and cytokinesis. It has also been established that the nuclear localization of two of these isoforms is essential for their function. The work presented here demonstrates that the higher eukaryotic CKIalpha isoform is also present within nuclei of certain established cell lines and associated with discrete nuclear structures. The nature of its nuclear localization was characterized. In this regard, CKIalpha was shown to colocalize with factors involved in pre-mRNA splicing at nuclear speckles and that its association with these structures exhibited several biochemical properties in common with known splicing factors. The kinase was also shown to be associated with a complex that contained certain splicing factors. Finally, in vitro, CKIalpha was shown to be capable of phosphorylating particular splicing factors within a region rich in serine/arginine dipeptide repeat motifs suggesting that it has both the opportunity and the capacity to regulate one or more steps of mRNA metabolism.