Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

Isolation and characterization of fatty acid-binding protein from rat brain

A fatty acid-binding protein has been identified and isolated from the cytosol fraction of rat brain. The fatty acid-binding protein was purified to homogeneity by gel filtration and preparative isoelectric focusing. The binding protein was different from Z protein from rat liver in its isoelectric point and immunological reactivity, in spite of its similar molecular weight of 12,000. Rabbit antibodies against rat liver Z protein were used to demonstrate that the fatty acid-binding proteins from rat liver and brain are immunologically unrelated, and that no Z protein is present in rat brain cytosol.     

A 14-kilodalton selenium-binding protein in mouse liver is fatty acid-binding protein

In a previous study, we purified three selenium-binding proteins (molecular masses 56, 14, and 12 kDa) from mouse liver using column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aim of the present study was to determine the amino acid sequence of the 14-kDa protein thereby establishing any relationship with known proteins. Although the amino terminus of the 14-kDa protein was blocked, separate in situ digestions of the protein with endoproteinases Glu-c and Lys-c gave overlapping peptides that provided a continuous sequence of 93 amino acids. This sequence exhibited a 92.5% sequence homology with rat liver fatty acid-binding protein. In situ enzymatic digestion and partial sequencing of a 12-kDa selenium-binding protein revealed identical homology to the 14-kDa protein. The 14-kDa protein bound specifically to an oleate-affinity column from which the protein and 75Se coeluted. Delipidation or sodium dodecyl sulfate treatment failed to remove 75Se from the protein, indicating that the selenium moiety was tightly bound to the protein. These observations confirm that the mouse liver selenium-binding 14-kDa protein is a fatty acid-binding protein. The nature of the selenium linkage to the protein still needs to be explored.     

A fatty acid-binding protein and a protein disulphide isomerase-related protein expressed in urochordate gonad cytosol

Despite the evolutionary-tree data suggesting that gene duplication leading to the divergence of the three branches which heart, liver and intestinal fatty acid-binding proteins belong to must have occurred before the vertebrate/invertebrate split, only the heart fatty acid-binding protein has been reported for invertebrates. In an attempt to shed light on this apparent inconsistency the presence of the other two branch members was investigated in the Urochordata Molgula pedunculata, an ascidian species close to vertebrates. The mantle-, gonad- and digestive tube-cytosolic fractions, obtained by centrifugation at 106,000 g, were incubated separately with [1-(14)C]palmitic acid and then fractionated on a Sephadex G-75 column. In the case of gonads and digestive tube, radioactive peaks corresponding to a molecular mass of 14-16 kDa, characteristic of fatty acid-binding proteins, were detected. When the experiment was performed on the mantle, this peak showing fatty acid binding capacity was absent. Western Blot of the radioactive 14-16 kDa Sephadex fraction from the urochordate gonad cross-reacted with rat liver fatty acid-binding protein anti-serum but did not do so with anti-rat intestinal, adipocyte or heart fatty acid-binding protein antisera. The material from the digestive tube was not recognized by any of the antisera. The most abundant protein in said 14-16 kDa fraction was a protein disulphide isomerase-related protein. Its partial amino acid sequence was determined.     

Disulfide bonds in rat cutaneous fatty acid-binding protein

Unlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid-binding proteins contain a large number of cysteine residues. The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and mass-spectrometric analyses. Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127, though extent of formation of the first disulfide bond was rather low in another preparation. Cys-43 was free cysteine. Homology modeling study of the protein indicated the close proximity of the sulfur atoms of these cysteine pairs, supporting the presence of the disulfide bonds. These disulfide bonds appear not to be directly involved in fatty acid-binding activity, because a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as examined by displacement of a fluorescent fatty acid analog by long-chain fatty acids. However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one between Cys-120 and Cys-127, and that fatty acid-binding proteins play multiple roles suggests that some functions of cutaneous fatty acid-binding protein might be regulated by the cellular redox state through formation and reduction of disulfide bonds. Although we cannot completely exclude the possibility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain disulfide bonds.     

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

Butyrate-binding protein from rat and mouse liver

A novel component which specifically binds butyrate was found in rat and mouse liver. This component, termed butyrate binding protein (BBP), is localized in the cytosolic fraction and exhibits protein characteristics, such as heat- and protease-sensitivity. The size of BBP was found to be 7.6S, while it was converted to subunits of 45,000--48,000 dalton by treatment with sodium dodecyl sulfate. The dissociation constant of the binding of BBP with butyrate was 2.22 X 10(-6) M in the standard assay. 30-Fold purification of BBP was achieved by batch-wise adsorption and elution from CM-cellulose and hydroxylapatite column chromatography. BBP is clearly distinguishable from the fatty acid-binding protein found previously on the basis of its size and binding specificity.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Identification by photoaffinity labeling of fatty acid-binding protein as a potential warfarin receptor in rat liver

Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.     

The binding of the fluorescent ATP analogue 2'(3')-trinitrophenyladenosine-5'-triphosphate to rat liver fatty acid-binding protein.

The less polar fluorescent analogue of ATP, 2'(3')-trinitrophenyl-5'-triphosphate bound to rat liver fatty acid-binding protein with high affinity (Kd 6.3 x 10(-6) M) and 1:1 molar stoichiometry. This probe bound to the fatty acid binding site of the protein and was displaced by oleic acid and oleoyl CoA. High concentrations of ATP did not cause significant displacement of the fluorescent ATP analogue. Since the anionic part of this molecule is the triphosphate group it is difficult to envisage this group being accommodated at an anion binding site within the non-polar core of this protein as is the case with other fatty acid binding proteins. Therefore it is anticipated that the ligand must bind to liver fatty acid-binding protein with this triphosphate group surface exposed. Caution must be exercised when using the more hydrophobic fluorescent analogue of ATP to investigate the ATP binding properties of proteins.     

Analysis of the fatty acid components in a perchloric acid-soluble protein

We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Mu?oz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.     

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.     

One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14–15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30–40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast

The chloroplastic isoform of glutamine synthetase (GS-2, EC 6.3.1.2) from Nicotiana tabacum L. is phosphorylated at the serine residues. At least three of the six GS-2 subunits separated by two-dimensional polyacrylamide gel electrophoresis cross-reacted with an antibody raised against phosphoserine. This provoked the question as to whether 14-3-3 proteins might be present in the chloroplast and bind to chloroplastic GS-2. Although two different 14-3-3 proteins of 32 and 30 kDa were present in total leaf extracts, in the soluble fraction of chloroplasts, only the 32-kDa 14-3-3 protein was immunodetected with an antibody raised against a conserved region of 14-3-3 protein from corn. This demonstrates the presence of a chloroplast-located isoform of 14-3-3 proteins in tobacco. To examine a putative binding of GS-2 to these 14-3-3 proteins in vivo, the native GS-2 holoenzyme was probed with a 14-3-3 antibody. The strong cross-reaction between GS-2 and the 14-3-3 antibody clearly points to a binding of GS-2 and 14-3-3 in tobacco chloroplasts. Only those oligomers of GS-2 that were strongly associated with 14-3-3 proteins were catalytically active.     

Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members.

The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salomé, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least partial identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions, since they were not disrupted by NS2 C-terminal modifications that impaired virus replication. Binding of NS2 to 14-3-3 proteins was detected in various cells of mouse, rat, hamster, monkey, and human origin, irrespective of NS2 dispensability and host cell transformation or permissiveness. The ubiquitous 14-3-3 proteins were recently reported to associate with several other cellular or viral polypeptides involved in signal transduction and/or cell cycle regulation pathways (A. Aitken, Trends Biochem. Sci. 20:95-97, 1995). The NS2 products may connect with one of these pathways through their interaction with specific 14-3-3 polypeptides.     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, 14-3-3eta, or 14-3-3zeta, respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834-21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cdelta (PKCdelta) based on the following observations. (i). Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an approximately 40-kDa protein band. This fraction had SDK1 activity approximately 50000-fold higher than that in the initial extract. (ii). This protein had approximately 53% sequence identity to the Ser/Thr kinase domain of PKCdelta based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii). A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the approximately 40-kDa band is the kinase domain of PKCdelta. The kinase activity of PKCdelta did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCdelta did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40-kDa kinase domain. PKCdelta and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.