Pituitary adenylate cyclase activating polypeptide anti-mitogenic signaling in cerebral cortical progenitors is regulated by p57Kip2-dependent CDK2 activity.
Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57Kip2-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27Kip1, p57Kip2) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G1/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57Kip2 protein, but not p21Cip1 or p27Kip1, suggesting that p57Kip2 mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57Kip2 association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57Kip2 interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex.
Carey
Rebecca G
RG
Department of Neuroscience and Cell Biology, Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Li
Baogang
B
DiCicco-Bloom
Emanuel
E
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000378:metabolism; D019070:Cell Lineage / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D050761:Cyclin-Dependent Kinase Inhibitor p57; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005314:Embryonic and Fetal Development / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D009479:Neuropeptides / Q000378:metabolism* / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C495401:ADCYAP1 protein, human; C495403:Adcyap1 protein, rat; C493964:CDKN1C protein, human; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050761:Cyclin-Dependent Kinase Inhibitor p57; D046911:Macromolecular Substances; D009479:Neuropeptides; D009687:Nuclear Proteins; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases