Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence.

Abstract

Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C1, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the y subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia cofi by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of the two sequences was sufficient for the phorbol ester binding. Conserved region C2, another region in the regulatory domain, was apparently needed for the enzyme to require Ca2' for phorbol ester binding activity.Protein kinase C (PKC) has attracted much attention since it plays a pivotal role in signal transduction for activation of many cellular functions and for control ofcell proliferation (1, 2 The C1 region has a tandem repeat characteristic of a sequence that resembles the cysteine-rich zinc-finger motif. Such a sequence motif has been identified in many DNA binding proteins that appear to be active in transcriptional regulation (14,15). However, there is presently no indication that PKC interacts with nucleic acids, and hence no information is available as to the function of this cysteine-rich sequence. By using various deletion and point mutants of the y subspecies of rat PKC expressed in Escherichia coli, the studies described herein will show that the cysteine-rich zinc-finger-like sequence is indispensable for the binding of phorbol ester to the enzyme. MATERIALS AND METHODSConstruction of Expression Plasmids. Each mutant was constructed as shown in Fig. 1. The cDNA fragment encoding amino acids 1-373 of the 'y subspecies of rat PKC was obtained from its cDNA clone of ACKRyl (10) by digestion with Nco I and BamHI. The cohesive ends of this fragment were altered to BamHI sites by E. coli DNA polymerase I (large fragment) and BamHI linkers (8 bases). It was cloned into the BamHI site of the T7 RNA polymerase-dependent expression vector pET3c (16,17), forming an in-frame fusion product with the first 11 amino acids of the T7 gene 10 product. This plasmid, designated pTB965, could express 389 amino acid residues because of the addition of 16 residues derived from T7 and linkers. Plasmid pTB967 was constructed by insertion of a BamHI-Sau3A fragment, encoding amino acids 4868The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A Protein Kinase Cδ-binding Protein SRBC Whose Expression Is Induced by Serum Starvation

“…In addition, the fact that SRBC binds to phosphatidylserine suggests that phosphatidylserine mediates or stabilizes the interaction between SRBC and PKC␦ through its bridging. Interaction with SRBC was also observed for MCD209, a deletion mutant of PKC␦ lacking the kinase domain and the C-terminal half of the cysteine-rich domain that is responsible for the binding to phorbol ester or diacylglycerol (32,33). This indicates that these functional domains are dispensable for the interaction with SRBC.…”

“…GST-SRBC and GST were also detected by Western blot analysis using anti-GST antibody (lanes 4 and 5). Overlay assay was performed in the presence (lanes 6 -8) or absence (lanes 9 -11) of phosphatidylserine using 32 protein) fusion proteins in E. coli (Fig. 4A) and used as probes for the overlay assay.…”

TaCHP: A Wheat Zinc Finger Protein Gene Down-Regulated by Abscisic Acid and Salinity Stress Plays a Positive Role in Stress Tolerance

“…DC1 domain-containing proteins have been implicated in plant response to abiotic stress, as for example the tobacco (Nicotiana tabacum) genes NtDC1A and NtDC1B that responded rapidly and strongly to AaGlucan, an elicitor of some stress-related genes (Shinya et al, 2007). DC1 domain, which is able to bind diacylglycerol (DAG) and phorbol esters (an analog of DAG) in a phospholipid and zinc-dependent fashion, presents in the N-terminal region of protein kinase C (PKC), a family of Ser/Thr protein kinases (Ono et al, 1989). DAG activates PKC (Azzi et al, 1992) and acts as a signal transducer in a Ca 2+ -dependent manner.…”

Immunocharacterization of β‐ and ζ‐subspecies of protein kinase C in bovine neutrophils

“…The other PKC isozyme of bovine neutrophil reacts with the anti-~'-antiserum and is present in relatively low amounts as compared to the ~-isoenzyme. So far, the ~'-PKC isozyme has been identified in expression experiments using the corresponding specific cDNA [13]. Only recently, ~'-PKC was described in nuclei of nerve cells [14].…”