Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C

We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.     

Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3

Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. Here, we show that the endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. Numb binds to integrin-betas and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, we propose a model in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge.     

A mass conserved reaction-diffusion system captures properties of cell polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5'-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction-diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction-diffusion system with diffusion-driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction-diffusion system with diffusion-driven instability is one of such principles of cell polarity.     

A Mass Conserved Reaction–Diffusion System Captures Properties of Cell Polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5′-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction–diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction–diffusion system with diffusion–driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction–diffusion system with diffusion-driven instability is one of such principles of cell polarity.     

Neuronal polarity: from extracellular signals to intracellular mechanisms

After they are born and differentiate, neurons break their previous symmetry, dramatically change their shape, and establish two structurally and functionally distinct compartments - axons and dendrites - within one cell. How do neurons develop their morphologically and molecularly distinct compartments? Recent studies have implicated several signalling pathways evoked by extracellular signals as having essential roles in a number of aspects of neuronal polarization.     

PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity

The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end–tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation.     

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr¹⁹⁹ by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr¹⁹⁹ remains at centrosomes. It has been shown that Thr¹⁹⁹ phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr¹⁹⁹. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr¹⁹⁹-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.     

Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway

The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3beta (GSK-3beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3beta. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway and mitogen-activated protein kinase cascade.     

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.