Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P 2 and PI-3,4,5-P 3 in the plasma membrane. A major advance in our understanding of signal transduction pathways has come from the realization that many signaling proteins possess one or more self-contained domains that mediate important regulatory interactions with other cellular structures. Many of these domains, such as Src homology domains 2 and 3 (SH2 and SH3 domains), were initially identified as mediators of protein-protein interactions, but it is now apparent that some domains are also involved in high-affinity binding to certain modified phospholipids. Just as SH2 domains have been demonstrated to mediate a regulatable, reversible association between two proteins, depending on the presence or absence of a phosphate group on key tyrosine residues, so it has recently become clear that pleckstrin homology (PH) domains can mediate a similar association with the plasma membrane, depending on the presence or absence of phosphate on the D3 position of the myo-inositol ring of phosphatidylinositides (PI) (6,12,30,44,63). This property of PH domains forms the basis for a regulatable, reversible association of PH domain-containing proteins with the phosphoinositide-rich regions of the plasma membrane, an event that plays an important role in regulating the activities of several enzymes important in signaling pathways.The importance of PH domain-mediated interactions with the plasma membrane is well illustrated by the mechanisms of activation of the ubiquitous serine/threonine kinase Akt (also known as protein kinase B) and the B-cell and mast cell protein tyrosine kinase (PTK) Btk. Both kinases possess PH domains within their amino termini. Akt plays an important role in growth control and protection from apoptosis, and is activated only when sufficient levels of both PI-3,4-P 2 and PI-3,4,5-P 3 are present in the plasma membrane (10,12,16,18). The mechanism of activation involves PH domain-dependent corecruitment of Akt and the Akt kinase PDK1 (which also contains a PH domain) to the plasma membrane by high-affinity interactions with PI-3,4-P 2 and PI-3,4,5-P 3 , respectively. Colocalized PDK1 phosphorylates Akt on Thr-308, catalyzing autophosphorylation on Ser-473, and activating the kinase (2, 71). The Btk kinase plays a vital ...

The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a -/- mouse that recapitulates features of Meckel syndrome including embryonic lethality and multi-organ defects. Cilia are absent in Cc2d2a -/- embryonic node and other somatic tissues; disruption of cilia-dependent Shh signaling appears to underlie exencephaly in mutant embryos. The Cc2d2a -/- mouse embryonic fibroblasts (MEFs) lack cilia though mother centriole and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a -/- MEFs, subdistal appendages are lacking or abnormal by transmission-EM. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.

The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/ 182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells. M ammalian sensory epithelia, such as those underlying vison, hearing, smell, and balance, consist of ciliated sensory receptor cells. Although highly specialized, similarities in embryonic origin underlie common features in their development (1). Following specification, lineage-restricted postmitotic precursors become structurally and functionally mature through a series of cellular differentiation events, collectively described as terminal differentiation (2). During maturation, sensory receptor cells typically develop microtubule-based primary cilia and in some cases actin-based membrane protrusions on apical membranes, which are sensory organelles, while maintaining apical basal polarity within sensory epithelia. In photoreceptors, for example, the extension of outer segments (OSs), specialized sensory cilia, is central to postmitotic differentiation and necessary for light sensitivity (3). In auditory and vestibular hair cells, the proper formation of hair bundles is indispensable for detecting sound and head positions (4). Similarly, olfactory sensory neurons (OSNs), the odorant receptor cells in the olfactory epithelium, project multiple dendritic cilia into the mucous membrane of the nasal epithelium where olfactory signaling is initiated (5). Mature sensory epithelia are highly structured with specific spatial organization that is tied to their functions. Synchronized planar cell polarity (PCP) of hair cells in the inner ear, for example, provides their directional sensitivity (6), whereas the development of laminar architecture in the retina restricts photoreceptors to proper compartments for efficient wiring with secondary neuro...

Development of axons and dendrites constitutes a critical event in neuronal maturation and seems to require signaling through the planar cell polarity (PCP) pathway. Mutations in components of the PCP pathway lead to a spectrum of neurological phenotypes and disorders. For example, a missense mutation in Prickle 1 (Pk1) is associated with progressive myoclonus epilepsy (PME) in humans, and its reduced gene dosage increases sensitivity to induced seizure in mice. In an effort to unravel the role of the PCP pathway in mammalian neuronal development, we examined the expression of Pk1 in the central nervous system (CNS) using in situ hybridization (ISH) in combination with a genetic knock-in approach. We show that Pk1 transcripts are detected in the postmitotic cells of the subplate and cortical plate during mid- and late stages of cortical neurogenesis. In adult brain, Pk1 is expressed in distinct neuronal and glial cell populations, with dynamic formation of dendrites and glial processes during development. Of all the cell types in the mature retina, the highest expression of Pk1 is detected in cholinergic amacrine neurons. Knockdown of Pk1 by shRNA or dominant-negative constructs causes reduced axonal and dendritic extension in hippocampal neurons. Similarly, Pk1 knockdown in neonatal retina leads to defects in inner and outer segments and axon terminals of photoreceptors. Our studies implicate Pk1 function in axonal-dendritic development associated with the maturation of CNS neurons.