Cloning and characterization of p10, an alternatively spliced form of p15 cyclin-dependent kinase inhibitor.
We have cloned an alternatively spliced form of cyclin-dependent kinase (CDK) inhibitor p15 from human placenta. The alternative splice arises from an alternative 5' donor site in intron 1. An in-frame stop codon within the new exon, called exon 1beta, leads to translation of a Mr 10,000 protein identical to the NH2 terminus of p15 but contains a novel, basic COOH terminus. The alternatively spliced form, termed here as p10, is ubiquitously expressed in normal and tumor cell lines as shown by Northern hybridization and reverse transcription-PCR. Transforming growth factor beta1 induces the expression of p10 similarly to p15 in human HaCaT keratinocytes. Expression and analysis of p15 and epitope-tagged p10 in cells by immunohistochemistry showed similar localization of both to the cytoplasm and nucleus in mink epithelial cells and cytoplasmic localization in mouse fibroblasts. Analysis of the effects of p10 and p15 on cell growth indicated that both were transiently growth inhibitory in Mv1Lu and NIH 3T3 cells, but their stable expression did not significantly reduce the number of cell colonies. In contrast to p15, CDK4 and CDK6 did not coimmunoprecipitate p10 in transient expression assays in COS-7 cells. Furthermore, overexpression of p10 together with p15 in COS-7 cells did not interfere with the complex formation of p15 with CDK4 or CDK6. Thus, in the absence of detectable CDK binding, p10 is transiently able to restrain cell cycling, indicating that the alternative splicing of the CDK inhibitors presents further complexity in their regulation and functions.
Tsubari
M
M
Haartman Institute, Department of Virology, University of Helsinki, Finland.
Tiihonen
E
E
Laiho
M
M
D016475:3T3 Cells; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000502:physiology; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D002352:Carrier Proteins; C493968:Cdkn2b protein, mouse; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; C495902:Cdk4 protein, mouse; C495912:Cdk6 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases