The 33-kDa C-Terminal Domain of Raf-1 Protein Kinase Exhibits a RAS-Independent Serum- and Phorbol Ester-Induced Shift in Gel Mobility

Oláh, Zoltán; Ferrier, A.; Lehel, Csaba; Anderson, Wayne B.

doi:10.1006/bbrc.1995.2293

Cited by 10 publications

(7 citation statements)

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“…These data demonstrated that the C1 region of PKC⑀ is sufficient for interaction with the Btk PH domain. Neither RI-⑀ nor RIII-⑀ fragments derived from the regulatory and catalytic domains, respectively, of c-Raf-1 (38) bound to the PH domain (data not shown). RI-⑀ contains the zinc finger-like sequence of c-Raf-1 that has a low level homology (ϳ30% identity) with that of the C1 region of PKC.…”

Section: The Ph Domain Of Btk Interacts With Pure Preparations Of Pkcmentioning

confidence: 93%

Interactions between Protein Kinase C and Pleckstrin Homology Domains

Yao

Suzuki

Ozawa

et al. 1997

Journal of Biological Chemistry

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Pleckstrin homology (PH) domains comprised of loosely conserved sequences of ϳ100 amino acid residues are a functional protein motif found in many signal-transducing and cytoskeletal proteins. We recently demonstrated that the PH domains of Tec family protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk) interact with protein kinase C (PKC) and that PKC down-regulates Btk by phosphorylation. In this study we have characterized the PKC-BtkPH domain interaction in detail. Using pure PKC preparations, it was shown that the Btk PH domain interacts with PKC with high affinity (K D ‫؍‬ 39 nM). Unlike other tested phospholipids, phosphatidylinositol 4,5-bisphosphate, which binds to several PH domains, competed with PKC for binding to the PH domain apparently because their binding sites on the amino-terminal portion of the PH domains overlap. The minimal PKC-binding sequence within the Btk PH domain was found to correspond roughly to the second and third ␤-sheets of the PH domains of known tertiary structures. On the other hand, the C1 regulatory region of PKC⑀ containing the pseudosubstrate and zinc finger-like sequences was found to be sufficient for strong binding to the Btk PH domain. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC that interacts with the C1 region of PKC, inhibited the PKC-PH domain interaction, whereas the bioinactive PMA (4-␣-PMA) was ineffective. The isoform of PKC, which has a single zinc finger-like motif instead of the two tandem zinc finger-like sequences present in conventional and novel PKC isoforms, does not bind PMA. Thus, as expected, PH domain binding with PKC was not interfered with by PMA. Further, inhibitors that are known to attack the catalytic domains of serine/threonine kinases did not affect this PKC-PH domain interaction. In contrast, the presence of physiological concentrations of Ca 2؉ induced less than a 2-fold increase in PKC-PH domain binding. These results indicate that PKC binding to PH domains involve the ␤2-␤3 region of the Btk PH domain and the C1 region of PKC, and agents that interact with either of these regions (i.e. phosphatidylinositol 4,5-bisphosphate binding to the PH domain and PMA binding to the C1 region of PKC) might act to regulate PKC-PH domain binding.

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Section: The Ph Domain Of Btk Interacts With Pure Preparations Of Pkcmentioning

confidence: 93%

Interactions between Protein Kinase C and Pleckstrin Homology Domains

Yao

Suzuki

Ozawa

et al. 1997

Journal of Biological Chemistry

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“…While the metabolic labeling experiments clearly demonstrated the incorporation of phosphate into RIII-⑀ as a correlate of the mobility shift, the phosphate group could comprise part of a more complex modification rather than phosphorylation per se. Indeed, some investigators have found no effect by either phosphoserine-or phosphotyrosine-specific phosphatases on Raf-1 enzymatic activity (17,29,60). In contrast, more recent studies have shown that treatment of Raf-1 with membrane-associated protein phosphatases can inactivate its kinase activity (47).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, we have shown that the region of Raf-1 responsible for the serum and phorbol ester-induced shift in gel mobility is localized to a catalytically inactive COOH-terminal fragment of Raf-1 (29). Since the amino-terminal domain of Raf-1 is responsible for direct binding to Ras (14), these results suggested that the modification of Raf-1 can take place in the absence of direct interaction with Ras.…”

mentioning

confidence: 81%

“…Cells-Previously, we identified a 33-kDa catalytically inactive COOH-terminal fragment of Raf-1 (amino acids 381-643; designated RIII-⑀), which exhibited a Ras-independent serum-and phorbol ester-induced shift in gel mobility that mimicked the shift observed with full-length Raf-1 (29). Recent interest has focused on the possible role(s) of oxygen free radicals, including hydrogen peroxide (H 2 O 2 ), in the regulation of various signal transduction processes.…”

Section: Mobility Shift Of a Cooh-terminal Raf-1 Fragment In Mammaliamentioning

confidence: 99%

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Sequential Modification of Serines 621 and 624 in the Raf-1 Carboxyl Terminus Produces Alterations in Its Electrophoretic Mobility

Ferrier

Lee

Anderson

et al. 1997

Journal of Biological Chemistry

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The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H 2 O 2 ) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566 -643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.Raf-1, the product of the c-raf-1 protooncogene, is a cytoplasmic serine/threonine protein kinase that is activated in response to various mitogenic signals (1, 2). Activation of Raf-1 results in a cascade of events involving the phosphorylation and subsequent activation of additional proteins, including mitogen-activated protein (MAP) 1 kinase kinase (MEK) and MAP kinase, which ultimately lead to transcriptional activation and mitogenesis (3, 4). Thus the Raf-1 protein kinase plays a central role in cellular signal transduction and is a key player in transmitting signals from the plasma membrane to the nucleus (5, 6).The amino terminus of Raf-1 serves as a regulatory domain, while the catalytic domain resides in the carboxyl terminus, which also may contain regulatory elements (7). Earlier studies showed that the activation of Raf-1 by membrane-associated, protein-tyrosine kinases was dependent on cellular Ras activity (8 -...

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“…NIH3T3/DN-PKC⑀ cells overexpressing the kinase-inactive mutant of PKC⑀ were constructed by converting lysine 437 within the catalytic domain to an arginine and then cloned into the epitope-tagging vector p⑀MTH, as described previously (24). NIH3T3/BXB Raf cells overexpressing the N-terminally truncated, activated Raf-1 were produced by cloning into the p⑀MTH vector (25).…”

Section: Cell Linesmentioning

confidence: 99%