Gerald Radziwill

CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation

Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.

Mitotic Raf-1 Is Stimulated Independently of Ras and Is Active in the Cytoplasm

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.

Regulation of c-Src by binding to the PDZ domain of AF-6

c‐Src is a tightly regulated non‐receptor tyrosine kinase. We describe the C‐terminus of c‐Src as a ligand for a PDZ (postsynaptic density 95, PSD‐95; discs large, Dlg; zonula occludens‐1, ZO‐1) domain. The C‐terminal residue Leu of c‐Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c‐Src regulation in cells. As a candidate PDZ protein, we analysed AF‐6, a junctional adhesion protein. The AF‐6 PDZ domain restricts the number of c‐Src substrates, whereas knockdown of AF‐6 has the opposite effect. Binding of c‐Src to the AF‐6 PDZ domain interferes with phosphorylation of c‐Src at Tyr527 by the C‐terminal kinase, and reduces c‐Src autophosphorylation at Tyr416, resulting in a moderately activated c‐Src kinase. Unphosphorylated Tyr527 allows binding of c‐Src to AF‐6. This can be overcome by overexpression of CSK or strong activation of c‐Src. c‐Src is recruited by AF‐6 to cell–cell contact sites, suggesting that c‐Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c‐Src regulation by interaction with a PDZ protein.

c-Src is a PDZ interaction partner and substrate of the E3 ubiquitin ligase Ligand-of-Numb protein X1.

The very C‐terminus of c‐Src is a ligand for PDZ domains. In a screen for PDZ domains that interact with c‐Src, we identified one of the PDZ domains of the Ligand‐of‐Numb protein X1 (LNX1), a multiple PDZ domain scaffold and RING type E3 ubiquitin ligase. We demonstrate that the interaction of c‐Src with LNX1 depends on the C‐terminal PDZ ligand of c‐Src. Furthermore, we show that c‐Src phosphorylates LNX1. Moreover, c‐Src itself is ubiquitinated by LNX1, suggesting an interdependent regulation of c‐Src and LNX1.

RIP2 mediates LPS-induced p38 and IκBα signaling including IL-12 p40 expression in human monocyte-derived dendritic cells

IL‐12, the critical factor for the generation of the Th1 type immune response, is produced by dendritic cells (DC) upon stimulation with LPS. Different signal pathways mediate LPS‐induced expression of IL‐12 and involve PI3K, MAPK and the transcription factor NF‐κB. Here, we show that the kinase Raf is involved in the expression of IL‐12 in human DC stimulated by LPS. We demonstrate that Raf regulates the expression of the IL‐12 subunit p40 not via the kinase MEK, the major effector of Raf in growth factor‐dependent signaling, but via the receptor‐interacting protein 2 (RIP2) using specific inhibitors for MAPK pathways. RIP2 is a kinase participating in LPS/Toll‐like receptor 4 signaling. Knockdown of RIP2 by siRNA inhibited LPS‐dependent expression of IL‐12 p40. In addition, knockdown of RIP2 reduced phosphorylation of p38 MAPK, ERK and IκBα, which are known upstream regulators of IL‐12 production. Thus, in human DC LPS stimulates a signal cascade that involves the Raf‐dependent activation of RIP2 leading to expression of IL‐12 p40.

Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin‐1 at epithelial adherens junctions. Nectin‐1 serves as a mediator of cell‐to‐cell spread for Herpes simplex virus 1 (HSV‐1). We analyzed the role of AF6 protein in the viral spread and nectin‐1 clustering at cell–cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV‐1 spreading, however, the clustering of nectin‐1 at cell–cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV‐1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E‐cadherin‐dependent cell adhesion.

JNK MAP Kinase

The c-Jun NH2-terminal kinase (JNK) is a member of the MAPK family and is activated in response to cellular stress, such as inflammatory cytokines, heat shock, and UV-light …

ERK MAP Kinase

The ERK MAP kinases, ERK1 and ERK2, are the downstream kinases of a signal cascade composed of the MAPKs ERK1/2, the MAPKKs MEK1/2, and the MAPKKKs of the Raf family …

p38 MAP Kinase

P38 MAP kinases are the downstream elements of specific MAP kinase pathways, which are activated by cellular stress stimuli such as cytokines, UV-light, heat shock, osmotic stress, and bacterial pathogens …