Mutational Analysis of the Regulatory Mechanism of PKN: The Regulatory Region of PKN Contains an Arachidonic Acid-Sensitive Autoinhibitory Domain

Yoshinaga, Chiho; Mukai, Hideyuki; Toshimori, Masanao; Miyamoto, Masaaki; Ono, Yoshitaka

doi:10.1093/oxfordjournals.jbchem.a022476

Cited by 58 publications

(90 citation statements)

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“…The first of these repeats, HR1a, is now known to incorporate the inhibitory pseudo-substrate site (17). PRK1/2 kinase activity is enhanced by binding of the small GTP-binding proteins Rho and Rac in a GTP-dependent manner (18 -21), by binding of fatty acids such as arachidonic acid (22,23), and by the removal of the N-terminal regulatory domain from the catalytic domain by caspase 3 cleavage during apoptosis (12,24). Progress has been made recently in the dissection of the regulatory mechanisms employed by the Rho family effector molecules PAK (25) and Wiskott-Aldrich syndrome protein (26).…”

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confidence: 99%

Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

Owen

Lowe

Nietlispach

et al. 2003

Journal of Biological Chemistry

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PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix ␣5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.

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confidence: 99%

Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

Owen

Lowe

Nietlispach

et al. 2003

Journal of Biological Chemistry

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“…Importantly, Rho-GTP binding activates PKN (45,46). Interestingly, PKN is also possibly regulated by autophosphorylation and binding to lipids, such as arachidonic acid (43,63,64), and is negatively regulated by intramolecular binding of a putative pseudosubstrate in the NH 2 -terminal region of catalytic domain. Given the ability of multiple modalities to regulate PKN, it is conceivable that E6 binding may achieve a positive or negative regulation of its activity.…”

Section: Discussionmentioning

confidence: 99%

PKN Binds and Phosphorylates Human Papillomavirus E6 Oncoprotein

Gao¹,

Kumar²,

Srinivasan³

et al. 2000

Journal of Biological Chemistry

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The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two viral oncoproteins E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary epithelial cells has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid-and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.

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“…Recombinant viruses encoding glutathione S-transferase (GST)͞PKN (543-942) (the constitutively active form of human PKN) and GST͞PKN (543-942)-K644E (the kinase-negative form of PKN) have been described (23). The recombinant viruses for GST͞Wee1 (full-length Xenopus Wee1), GST͞cyclin B1 (the indestructible form of Xenopus cyclin B1), and histidine (His) 6 ͞Cdc2 (N133A) (the kinase-negative form of Xenopus Cdc2) were provided by M. Iwabuchi (Tokyo Institute of Technology, Yokohama, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant viruses for GST͞Wee1 (full-length Xenopus Wee1), GST͞cyclin B1 (the indestructible form of Xenopus cyclin B1), and histidine (His) 6 ͞Cdc2 (N133A) (the kinase-negative form of Xenopus Cdc2) were provided by M. Iwabuchi (Tokyo Institute of Technology, Yokohama, Japan). Recombinant virus encoding GST͞PKC (386 -737) (the constitutively active form of rat PKC) was generated by using the baculovirus transfer vector of pBlueBacHis͞GST (23) and the cDNA fragment encoding the catalytic domain of rat PKC (24). Recombinant virus encoding GST͞Cdc25C was generated by using the baculovirus transfer vector of pBlueBacHis͞GST and the full-length Xenopus Cdc25C cDNA (25) provided by N. Nakajo (Kyushu University, Fukuoka, Japan).…”

Section: Methodsmentioning

confidence: 99%

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PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

Misaki¹

2000

Proceedings of the National Academy of Sciences

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The role of PKN, a fatty acid-and Rho small GTPase-activated protein kinase, in cell-cycle regulation was analyzed. Microinjection of the active form of PKN into a Xenopus embryo caused cleavage arrest, whereas normal cell division proceeded in the control embryo microinjected with buffer or the inactive form of PKN. Exogenous addition of the active form of PKN delayed mitotic timing in Xenopus egg cycling extracts judging by morphology of sperm nuclei and Cdc2͞cyclin B histone H1 kinase activity. The kinase-negative form of PKN did not affect the timing, suggesting that delayed mitotic timing depends on the kinase activity of PKN. The dephosphorylation of Tyr-15 of Cdc2 was also delayed in correlation with Cdc2͞cyclin B histone H1 kinase activation in extracts containing active PKN. The Cdc25C activity for the dephosphorylation of Tyr-15 in Cdc2 was suppressed by pretreatment with the active form of PKN. Furthermore, PKN efficiently phosphorylated Cdc25C in vitro, indicating that PKN directly inhibits Cdc25C activity by phosphorylation. These results suggest that PKN plays a significant role in the control of mitotic timing by inhibition of Cdc25C. P rotein phosphorylation͞dephosphorylation reaction is a key event in the regulation of cell division. In dividing eukaryotic cells, entry into mitosis is governed by the M phase-promoting factor. This factor consists of the Cdc2 protein kinase and cyclin B and acts by phosphorylating substrates that are essential for the execution of mitotic processes (1). Before mitosis, the activity of Cdc2͞cyclin B histone H1 kinase is suppressed through inhibitory phosphorylation of Tyr-15 and Thr-14 residues of Cdc2 by the Wee1 and Myt1 kinases (2-5). At onset of mitosis, the phosphatase Cdc25C removes these inhibitory phosphate groups from Cdc2 and thereby activates Cdc2͞cyclin B histone H1 kinase. The activity of Cdc25C is strictly regulated, probably by phosphorylation, and is low during interphase and high at mitosis (6, 7). Thus, entry into mitosis is under the control of a tightly regulated network of protein kinases and phosphatases. Although upstream players such as Chk1 and Cds1 (8-10) in the tyrosine phosphorylation͞dephosphorylation of Cdc2 have been identified, it remains unclear how these enzymes are controlled during the cell cycle. There is a possibility that the unidentified protein kinase or kinases might participate in upstream regulation.PKN is a serine͞threonine protein kinase that has a catalytic domain highly homologous to protein kinase C (PKC) in the carboxyl-terminal region and a unique regulatory domain in the amino-terminal region (11-13). The amino-terminal region of PKN contains three repeats of a leucine zipper-like motif, and its kinase activity is stimulated by fatty acids such as arachidonic acid (13). We reported that PKN translocates from the cytosol to the nucleus of fibroblasts on exposure to stress such as heat shock and serum starvation (14) and that PKN is cleaved during apoptosis, presumably by caspase-3, which generates a constitutively...

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Mutational Analysis of the Regulatory Mechanism of PKN: The Regulatory Region of PKN Contains an Arachidonic Acid-Sensitive Autoinhibitory Domain

Cited by 58 publications

References 0 publications

Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)

PKN Binds and Phosphorylates Human Papillomavirus E6 Oncoprotein

PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

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