Interaction of integrin beta1 with cytokeratin 1 in neuroblastoma NMB7 cells.

Cytokeratin 1, an intermediate filament keratin, was isolated as a partner of the tyrosine kinase Src from neuroblastoma NMB7 cells. The cytokeratin 1-Src complex was found to be associated with the molecular scaffolder RACK1 (receptor for activated protein kinase C). Interestingly, the cytokeratin 1-Src-RACK1 complex was found to actively bind with membrane receptors such as integrin beta1. We are interested in using this complex to find downstream kinases and phosphatases that bind upon cytokine stimulation, especially during neurogenesis.

GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphate but not mammalian GAP.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.

Disrupting the transforming activity of shrimp ras(Q(61)K) by deleting the CAAX box at the C-terminus.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus. Ras transcription and protein levels had increased significantly in the cells transfected with the S-ras plasmid, compared to cells transfected with a control plasmid pcDNA3.1. The bacterially expressed GTP-locked S-Ras(Q(61)K) is successfully prenylated by rat protein geranylgeranyltransferase I (PGGTase I) and then polymerized with tubulin, in agreement with findings for GTP-locked mammalian K(B)-Ras(Q(61)K) in vitro. Shrimp protein farnesyltransferase (PFTase) of shrimp did not prenylate the GTP-locked shrimp S-Ras(Q(61)K) (Lin and Chuang. 1998. J Exp Zool 281:565-573), whereas rat PFTase efficiently catalyzed the farnesylation of GTP-locked S-Ras(Q(61)K). To investigate the effect of geranylgeranylation on cellular transformation, we generated S-ras(Q(61)K) mutants with deletion of the CAAX box [S-ras(Q(61)K)(-caax)] or replacement of the CAAX box [S-ras(Q(61)K)(Kcaax)] or replacement of the arginine-rich domain [S-ras(Q(61)K)(K-Lys)] with corresponding sequences from rat K(B)-ras(Q(61)K). BALB/3T3 cells transfected with DNA encoding S-ras(Q(61)K), S-ras(Q(61)K)(KCAAX), S-ras(Q(61)K)(K-Lys) were transformed successfully, but S-ras(Q(61)K)(-CAAX) was defective in its ability to transform. Thus, prenylation at CAAX is required for transformation. Either the geranylgeranylated or the farnesylated S-Ras(Q(61)K) was endowed with abilities to transform. The arginine-rich region in S-Ras or the lysine-rich clusters from the rat K(B)-Ras appear not essential for activity to transform.

Interaction of shrimp ras protein with mammalian caveolin-1.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang and Chuang. 1999. J Exp Zool 283:510-521). The caveolin-1 in the membrane fraction extractable with 2% octyl glucoside was significant reduced, compared to untransformed cells. To understand this in more detail, the interaction of S-Ras with caveolin was investigated using caveolin-1 purified from rat lungs. The purified caveolin-1 binds c-Src, suppressing its autophosphorylation. It also binds to phosphatidylserine-cholesterol liposomes. These reconstituted caveolin-phosphatidylserine-cholesterol vesicles, which act as a model of caveolae, recruit both bacterially expressed S-Ras and rat K(B)-Ras proteins, as demonstrated on western blots with antibodies against caveolin-1 and Ras. Caveolin-1 suppressed the intrinsic GTPase activity of S-Ras, sustaining it in the active GTP bound form. By contrast, caveolin-1 enhanced the intrinsic GTPase activity of K(B)-Ras, to convert it into the inactive GDP-bound form. These events suggest that caveolin may act as a docking site for Ras proteins and may be able to either maintain or alter their activity state. These events may be associated with the ability of S-ras(Q(61)K) to successfully transform cells.

Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda).

Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras.

Disrupting the geranylgeranylation at the C-termini of the shrimp Ras by depriving guanine nucleotide binding at the N-terminal.

In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian K(B)-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510-521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant S-Ras proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565-573) was unable to catalyze the transfer of [(3)H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441-449, 2000.

Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate.

A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.

Carboxy-terminal CFFL-sequence-specific monomeric protein geranylgeranyltransferase I from the eyes of the shrimp Penaeus japonicus.

Protein geranylgeranyltransferase I from the eyes of Penaeus japonicus geranylgeranylates predominantly the sequence CFFL and Drosophila-specific Ras1 carboxyl termini, with the sequence CKML, as well as mammalian-specific G gamma carboxyl termini, with the sequence CAIL, but not the protein farnesyltransferase-specific sequence CVLS. The purified protein geranylgeranyltransferase I from shrimp was evidenced by immunoblotting and polyacrylamide gel electrophoresis under denaturing conditions to consist of single subunit of Mr 66,000 +/- 500. Since the active protein geranylgeranyltransferase I was found to have a relative mass of 67,000 +/- 1,000, the purified enzyme was deduced to be a monomer. The enzyme had an optimal pH of 8.0 with 100 mM Tris as the buffer and a K(m) of 7 +/- 2 microM with the synthetic peptide KCFFL as the substrate. The enzyme was inhibited by Zn++ and Mg++ ions at micromolar concentrations.

Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding.

A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.

Carboxy-terminal CVLS-sequence-specific protein farnesyltransferase from the eyes of the shrimp Penaeus japonicus: purification and characterization.

Protein farnesyltransferase from the eyes of Penaeus japonicus farnesylates predominantly H-ras-specific carboxyl termini, with the sequence CVLS, but not the K-ras-specific sequence CVIM or the protein geranylgeranyltransferase-specific sequence CAIL. The purified protein farnesyltransferase from shrimp was found by immunoblotting and polyacrylamide gel electrophoresis under denaturing conditions to consist of subunits of Mr 49,000 and Mr 48,000. Since the active protein farnesyltransferase was found to have a relative mass of 100,000, the purified enzyme was deduced to be a heterodimer. The enzyme had an optimal pH of 6 and a K(m) of 14 +/- 1 microM with the synthetic peptide RTRCVLSH as the substrate. The enzyme was activated by Mn+2 and Mg+2 but inhibited by Ca+2 ions.

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea:Decapoda).

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M(r) 70,000 and M(r) 67,000, as well as less intense bands of proteins with M, 64,000 and M(r) 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M(r) 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl2, CuCl2 and Pb(NH3)3 at millimolar concentrations, but less inhibition was observed with CaCl2.

Purification of the delta isoenzyme of protein kinase C from the hepatopancreas of the shrimp Penaeus monodon with phosphorylation on tyrosine residues.

The delta isoenzyme of protein kinase C (PKC-delta), purified from the plasma membrane of the hepatopancreas of the shrimp Penaeus monodon is specifically phosphorylated at tyrosine residues, as demonstrated by specific dephosphorylation by phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. The specific activity of purified PKC-delta was 200 units/mg of protein. The subunits of M(r) 66,000, 62,000, and 58,000 of PKC-delta were not autophosphorylated after the addition of phosphatidylserine and diolein. However, the purified PKC-delta was active and catalyzed the phosphorylation of myelin basic protein. The kinase activity of the purified PKC-delta could be decreased after treatment with phosphotyrosyl protein phosphatase.

The zeta protein kinase C isoform from the testis of the grey mullet Mugil cephalus with a specific reaction protein of M(r) 48,000 on oolemma.

The zeta protein kinase C isoform (PKC-zeta) was purified from the testis of the grey mullet Mugil cephalus and has relative masses (M(r)) of 65,000 and 63,000. The subunits of PKC-zeta from spermatozoa degenerated to M(r) 58,000 and 53,000 after continuous freezing and thawing. Proteins of M(r) 48,000 on the oolemma of the grey mullet Mugil cephalus were found to be the reaction proteins of the PKC-zeta from spermatozoa.

Characterization of insulin receptor from the muscle of the shrimp Penaeus japonicus (Crustacea: Decapoda).

The beta-subunit of the insulin receptor from the muscle of the shrimp Penaeus japonicus exists as multiple subtypes with M(r) of 79,000, 77,000 and 75,000. Only the subunit of M(r) 79,000 is autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the human placenta. The detergent, Triton X-100, and the metal ion, Mn2+, caused a noticeable enhancement of the autophosphorylation of shrimp insulin receptors from the muscle. Okadaic acid activated the kinase activity of the insulin-stimulated insulin receptor, but not the basal activity of the insulin receptor without the addition of insulin. Further studies comparing the insulin binding of the shrimp insulin receptor in the regulation of kinase activity of the multiple beta-subunit subtypes from the shrimp muscle are under way.

The binding of corticosterone to the class-theta glutathione S-transferase from the eyes of the shrimp Penaeus japonicus (Crustacea: Decapoda).

The anionic homodimeric theta glutathione S-transferase (EC 2.5.1.18), with specific activities of 4.6 units/mg with 1-chloro-2,4-dinitrobenzene as substrate, from the eyes of the shrimp Penaeus japonicus, binds to corticosterone, which was confirmed by the analysis of the conjugate by SDS-PAGE and fluorography of proteins with bound (3)H-corticosterone.The reaction of the theta glutathione S-transferase with corticosterone was prevented by the presence of glutathione at 10 mM.

Specific phosphorylation of membrane proteins of Mr 44,000 and Mr 32,000 by the autophosphorylated insulin receptor from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda).

The insulin receptor, purified from the hepatopancreas of the shrimp Penaeus monodon, is a hydrophobic heterodimer of subunits with relative masses (Mr) of 70,000 and 58,000, as estimated by FPLC on Superose 12 and SDS-PAGE. Only the subunit of Mr 70,000 was autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. Proteins of Mr 44,000 and Mr 32,000 on the plasma membrane from the hepatopancreas of the shrimp Panaeus monodon were phosphorylated by the autophosphorylated insulin receptor from the shrimp hepatopancreas, but not by that from the human placenta. The detergent, Triton X-100, caused noticeable enhancement of the autophosphorylation of both shrimp and human insulin receptors.

Anionic glutathione S-transferases in shrimp eyes.

1. Two anionic isoenzymes of glutathione transferases (EC 2.5.1.18), QI and QII, have been purified from the eyes of the shrimp Penaeus japonicus by using a combination of S-hexylglutathione affinity column chromatography and Mono-Q fast protein liquid chromatography (f.p.l.c.). 2. Both QI and II glutathione S-transferases are homodimers. They show similarity in substrate specificities and pH optima, but not in isoelectric points. 3. QI is distinct from QII by anion-exchange f.p.l.c., reverse-phase h.p.l.c. chromatography and amino acid sequencing analysis. QI has N-terminal amino acid sequences homologous to mu glutathione S-transferase, whereas QII is homologous to theta glutathione S-transferases.

Characterization of phosphotyrosyl protein phosphatase fom the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda).

Phosphotyrosyl protein phosphatase, purified from the hepatopancreas of Panaeus japonicus, is a monomeric enzyme with a relative mass (Mr) of 28,000, as estimated by size-exclusion FPLC on a Superose 12. It has a hydrophobic domain and can be extracted with the detergent CHAPS.The specific activity of the purified enzyme was 9,800 units/mg of protein. The purified enzyme had an isoelectric point less than 4.6, and an optimal pH of 6.5 with either a synthetic peptide or autophosphorylated receptors for insulin as the substrate. The purified phosphotyrosyl protein phosphatase from shrimp hepatopancreas dephosphorylated human and shrimp receptors for insulin and was inactivated by ZnC1(2), LiC1, MgC1(2), and NaF.

Purification and characterization of acidic alpha-D-mannosidase from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda).

Acidic alpha-D-mannosidase purified from the hepatopancreas of Penaeus monodon is pentameric, with a Mr 251,000 as estimated by size-exclusion FPLC on a Superose 12, and retains a hydrophobic domain, being stable to heating at 65 degree C for 1 h. The specific activity of the purified enzyme is 1,923 units/mg of protein. After polyacrylamide gel electrophoresis under denaturing conditions, the purified acidic alpha-D-mannosidase from shrimp was found to consist of a single homogeneous type of monomer of Mr 51,000. The purified enzyme has an isoelectric point of 4.4 +/- 0.1 and becomes more alkaline after the removal of either sialic acid or phosphate groups. The apparent K(m), value of alpha-D-mannosidase from shrimp hepatopancreas with 4-methylumbelliferyl-alpha-D-mannopyranoside as substrate is 243 micrometer.

Purification and characterization of an alpha-glucosidase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: decapoda).

1. The alpha-glucosidase purified from the hepatopancreas of Penaeus japonicus is not membrane-bound and labile to heating at 65 degree C for 10 min. 2. The specific activity of the purified enzyme is 223 units/mg of protein. After polyacrylamide gel electrophoresis under denaturing conditions, the purified alpha-glucosidase from shrimp was found to consist of monomers of M(r) 105,000. 3.The alpha-glucosidase from shrimp has an isoelectric point (pI) of 3.8 and becomes more alkaline after the removal of sialic acid and phosphoric acid. 4.The shrimp enzyme has the pH optimum at 5.0 and its K(m) was 125 micrometer with 4-methylumbelliferyl-alpha-D-glucoside as substrate.