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Two species of human CRK cDNA encode proteins with distinct biological activities.

ABSTRACT: Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.

SUBMITTER: Matsuda M

PROVIDER: S-EPMC364597 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Two species of human CRK cDNA encode proteins with distinct biological activities.

Matsuda M M Tanaka S S Nagata S S Kojima A A Kurata T T Shibuya M M

Molecular and cellular biology 19920801 8

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse pro ...[more]

PMID: 1630456

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Project description:BackgroundNF-kappaB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-kappaB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain.Methods/principal findingsWe characterized two NF-kappaB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-kappaB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-kappaB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-kappaB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-kappaB proteins affect DNA binding as in human NF-kappaB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant.Conclusions/significanceThese results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-kappaB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.

Project description:Human saliva contains a single 72,000-M(r) species which specifically reacted with rabbit anti-[human placental folate receptor (PFR)] serum on SDS/PAGE and Western blots. Although a specific radioimmunoassay for human PFR and related folate-binding proteins (FBPs) identified 55 ng of cross-reacting material (CRM) per mg of crude salivary proteins, only a minor fraction (1.6 ng) specifically bound radiolabelled folate. The major fraction of CRM did not contain bound endogenous folate and did not bind radiolabelled folates. On the basis of folate binding, salivary CRM species to PFR were designated as either functional (f-FBP) or non-functional (nf-FBP) species respectively. nf-FBPs and f-FBPs were isolated by different purification schemes. Both purified f-FBPs and nf-FBPs migrated as a single apparent 72,000-M(r) species on SDS/PAGE, but on Sephacryl S-200 gel filtration and sucrose-density-gradient ultracentrifugation they were eluted/sedimented with 40,000-M(r) markers. Each microgram of purified f-FBP and nf-FBP was measured in the radioimmunoassay for PFR as being equivalent to 18 ng and 24 ng of CRM respectively, indicating low epitope-relatedness to PFR. The Kd of f-FBPs was 50 pM and 0.94 mol of folate was bound/mol of protein. f-FBPs exhibited an unusual dependence on Triton X-100 for optimal ligand binding, despite the fact that Triton X-100 micelle binding was not demonstrated. The relative order of affinity of f-FBPs for pteroylglutamate greater than methotrexate greater than 5-formyltetrahydrofolate greater than 5-methyltetrahydrofolate was also distinct from that of purified PFR. Whereas amino acid and carbohydrate analysis revealed that nf-FBP (M(r) 51,400) and f-FBP (M(r) 39,200) were distinct glycoproteins with 8 and 13% carbohydrate respectively, isoelectric focusing and immunological studies suggested some structural identity. The presence of f-FBP and nf-FBP in normal saliva raises new questions about their possible role in vivo.

Dataset Information

Two species of human CRK cDNA encode proteins with distinct biological activities.

Publications

Two species of human CRK cDNA encode proteins with distinct biological activities.

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