Cyclin-dependent kinase 4 (CDK4) is known to be a 33 kD protein that drives G1 phase progression of the cell cycle by binding to a CCND protein to phosphorylate RB proteins. Using different CDK4 antibodies in western blot, we detected 2 groups of proteins around 40 and 33 kD, respectively, in human and mouse cells; each group often appeared as a duplet or triplet of bands. Some CDK4 shRNAs could decrease the 33 kD wild-type (wt) CDK4 but increase some 40 kD proteins, whereas some other shRNAs had the opposite effects. Liquid chromatography-mass spectrometry/mass spectrometry analysis confirmed the existence of CDK4 isoforms smaller than 33 kD but failed to identify CDK4 at 40 kD. We cloned one CDK4 mRNA variant that lacks exon 2 and encodes a 26 kD protein without the first 74 amino acids of the wt CDK4, thus lacking the ATP binding sequence and the PISTVRE domain required for binding to CCND. Co-IP assay confirmed that this δ;E2 protein lost CCND1-and RB1-binding ability. Moreover, we found, surprisingly, that the wt CDK4 and the δE2 could inhibit G1-S progression, accelerate S-G2/M progression, and enhance or delay apoptosis in a cell line-specific manner in a situation where the cells were treated with a CDK4 inhibitor or the cells were serum-starved and then replenished. Hence, CDK4 seems to be expressed as multiple proteins that react differently to different CDK4 antibodies, respond differently to different shRNAs, and, in some situations have previously unrecognized functions at the S-G2/M phases of the cell cycle via mechanisms independent of binding to CCND and RB.
Bibliographical noteFunding Information:
We would like to thank Dr Chengguang Wang at Thomas Jefferson University for kindly providing us the CDK4−/−MEF and the CDK4 and CCND1 cDNAs, and thank Dr Lloyd Greene at Columbia University for providing us a CDK4 shRNA plasmid. We also want to thank Fred Bogott, MD, PhD, at Austin Medical Center, Austin, Minnesota, for his excellent English editing of this manuscript. This work was supported by a grant from the Department of Defense of United States (DOD Award W81XWH-11-1-0119) to DJ Liao.
- Alternative splicing
- Cell cycle