RACK1, a Protein Kinase C Scaffolding Protein, Interacts with the PH Domain of p120GAP

2001 ◽  
Vol 283 (4) ◽  
pp. 888-895 ◽  
Author(s):  
Jackie A. Koehler ◽  
Michael F. Moran
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Scaffolding Protein ◽  
Ph Domain ◽  
Kinase C

Stratifin, a keratinocyte specific 14–3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity

1995 ◽  
Vol 108 (11) ◽  
pp. 3569-3579
Author(s):  
E. Dellambra ◽  
M. Patrone ◽  
B. Sparatore ◽  
A. Negri ◽  
F. Ceciliani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Terminal Differentiation ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Database ◽  
Kinase C ◽  
Human Keratinocyte ◽  
Protein Kinase C Activity

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14–3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14–3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

Janus Kinases Interact with and Phosphorylate Rack-1 (Receptor for Activated C Kinase-1), a WD Motif Containing Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2184-2184
Author(s):  
Takashi Haro ◽  
Kazuya Shimoda ◽  
Haruko Kakumitsu ◽  
Kenjirou Kamezaki ◽  
Atsuhiko Numata ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
B Lymphocyte ◽  
Kinase Domain ◽  
Scaffolding Protein ◽  
Cytokine Signaling ◽  
Interacting Protein ◽  
Kinase C ◽  
Tyrosine Kinase 2 ◽  
Lymphocyte Growth

Abstract We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

scholarly journals The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration

2001 ◽  
Vol 114 (9) ◽  
pp. 1691-1698 ◽  
Author(s):  
C.S. Buensuceso ◽  
D. Woodside ◽  
J.L. Huff ◽  
G.E. Plopper ◽  
T.E. O'Toole
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Scaffolding Protein ◽  
Kinase C ◽  
Bidirectional Signaling ◽  
Pkc Isoforms ◽  
Dependent Cell

The scaffolding protein, Rack1, is a seven-WD-domain-containing protein that has been implicated in binding to integrin (β) subunit cytoplasmic domains and to members of two kinase families (src and protein kinase C, PKC) that mediate integrin bidirectional signaling. To explore the role of Rack1 in integrin function we have transfected this protein in Chinese hamster ovary (CHO) cells. We have observed no effect of Rack1 overexpression on inside-out signaling as the ligand binding properties of CHO cells also expressing constitutively active or inactive integrins were not affected. In contrast, we observed that cells stably or transiently overexpressing Rack1 had decreased migration compared to mock transfected cells. Stable Rack1 transfectants also demonstrated an increased number of actin stress fibers and focal contacts. These effects on motility and cytoskeletal organization did not appear to result from Rack1 inhibition of src function as downstream substrates of this kinase were phosphorylated normally. In addition, expression of an active src construct did not reverse the migratory deficit induced by Rack1 overexpression. On the other hand when we overexpressed a Rack1 variant with alanine substitutions in the putative PKC binding site in its third WD domain, we observed no deficit in migration. Thus the ability of Rack1 to bind, localize and stabilize PKC isoforms is likely to be involved in aspects of integrin outside-in signaling.

scholarly journals The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain

2004 ◽  
Vol 382 (3) ◽  
pp. 957-966 ◽  
Author(s):  
Shin-ichi IMAI ◽  
Masahiro KAI ◽  
Keiko YAMADA ◽  
Hideo KANOH ◽  
Fumio SAKANE
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Diacylglycerol Kinase ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Bioactive Lipids ◽  
Intact Cells ◽  
Kinase C

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKδ1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKδ1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKδ1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKδ1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKδ1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKδ1.

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