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Alternative titles; symbolsp27(KIP1)KIP1HGNC Approved Gene Symbol: CDKN1BCytogenetic location: 12p13.1 Genomic coordinates (GRCh38): 12:12,717,367-12,722,368...

Alternative titles; symbols

  • p27(KIP1)
  • KIP1

HGNC Approved Gene Symbol: CDKN1B

Cytogenetic location: 12p13.1 Genomic coordinates (GRCh38): 12:12,717,367-12,722,368 (from NCBI)

▼ Description
CDKN1B, or p27(KIP1), is a cyclin-dependent kinase inhibitor that blocks the cell cycle in the G0/G1 phase upon differentiation signals or cellular insult. CDKN1B also regulates cell motility and apoptosis (summary by Cuesta et al., 2009).

▼ Cloning and Expression
Using a cDNA probe amplified from the MV1Lu mink cell line to screen a kidney cDNA library, Polyak et al. (1994) cloned human CDKN1B, which they called KIP1. The deduced 198-amino acid protein has a calculated molecular mass of 22.3 kD. It has a 60-amino acid N-terminal domain that shares 44% identity with the corresponding region of CIP1/WAF1 (CDKN1A; 116899). It also has a C-terminal bipartite nuclear localization signal and a consensus CDC2 (CDK1; 116940) phosphorylation site. KIP1 shares about 90% amino acid identity with mink and mouse Kip1, with highest identity in the N-terminal domain. Northern blot analysis detected variable expression of a 2.5-kb transcript in all human tissues examined.

Stegmaier et al. (1995) studied loss of heterozygosity (LOH) in the region 12p13-p12 in acute lymphoblastic leukemia; this chromosomal region often shows deletion in such patients. In 15% of informative patients, there was evidence of LOH of the TEL locus (600618) which was not evident on cytogenetic analysis. Detailed examination of patients with LOH showed that the critically deleted region included a second candidate tumor suppressor gene, referred to by them as KIP1, which encodes the cyclin-dependent kinase inhibitor previously called p27 (Toyoshima and Hunter, 1994 and Polyak et al., 1994). Based on the STS content of TEL-positive YACs, Stegmaier et al. (1995) reported that KIP1 and TEL were in close proximity.

▼ Gene Function
Polyak et al. (1994) showed that recombinant mouse Kip1 inhibited phosphorylation of histone H1 (see 142709) by human cyclin A (see CCNA1; 604036)-CDK2 (116953), cyclin E (CCNE1; 123837)-CDK2, and cyclin B1 (CCNB1; 123836)-CDK2 complexes. Addition of Kip1 also inhibited phosphorylation of RB (614041) by cyclin E-CDK2, cyclin A-CDK2, and cyclin D2 (CCND2; 123833)-CDK4 (123829). The isolated N-terminal domain of Kip1 had similar inhibitory activity in these assays. Kip1 bound to preactivated cyclin E-CDK2 complexes and prevented phosphorylation and activation of CDK2 in A549 human lung carcinoma cells. Kip1 activity was lowest in S phase in MV1Lu cells, and Kip1 overexpression inhibited S phase entry. Kip1 mRNA content remained unchanged during the cell cycle, suggesting that Kip1 activity was controlled at a posttranscriptional level. Polyak et al. (1994) concluded that KIP1 can inhibit both CDK activation and the kinase activity of assembled and activated cyclin-CDK.

CDK activation requires association with cyclins and phosphorylation by CAK (CCNH; 601953) and leads to cell proliferation. Inhibition of cellular proliferation occurs upon association of CDK inhibitor (e.g., CDKN1B) with a cyclin-CDK complex. Sheaff et al. (1997) showed that expression of CCNE1-CDK2 at physiologic levels of ATP resulted in phosphorylation of CDKN1B at thr187, leading to elimination of CDKN1B from the cell and progression of the cell cycle from G1 to S phase. At low ATP levels, the inhibitory functions of CDKN1B were enhanced, thereby arresting cell proliferation.

Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic upregulation of cyclin A (see 604036)-associated CDK2 activity. Levkau et al. (1998) showed that in apoptotic cells the carboxyl-termini of the CDK inhibitors CDKN1A and CDKN1B are truncated by specific cleavage. The enzyme involved in this cleavage is CASP3 (600636) and/or a CASP3-like caspase. After cleavage, CDKN1A loses its nuclear localization sequence and exits the nucleus. Cleavage of CDKN1A and CDKN1B resulted in a substantial reduction in their association with nuclear cyclin-CDK2 complexes, leading to a dramatic induction of CDK2 activity. Dominant-negative CDK2, as well as a mutant CDKN1A resistant to caspase cleavage, partially suppressed apoptosis. These data suggested that CDK2 activation, through caspase-mediated cleavage of CDK inhibitors, may be instrumental in the execution of apoptosis following caspase activation.

High levels of p27(KIP1), present in quiescent (G0) cells, have been shown to decline upon mitogen induction (Sherr and Roberts, 1995). Braun-Dullaeus et al. (1999) explored the role of p27(KIP1) and other cell cycle proteins in mediating angiotensin II (see 106150)-induced vascular smooth muscle cell hypertrophy or hyperplasia. Angiotensin II treatment (100 nM) of quiescent vascular smooth muscle cells led to upregulation of the cell cycle regulatory proteins cyclin D1 (168461), CDK2, proliferating cell nuclear antigen (176740), and CDK1. Levels of p27(KIP1), however, remained high, and the activation of the G1-phase CDK2 was inhibited as the cells underwent hypertrophy. Angiotensin II stimulated an increase in [(3)H]thymidine incorporation and the percentage of S-phase cells in p27(KIP1) antisense oligodeoxynucleotide (ODN)-transfected cells but not in control ODN transfected cells. The authors concluded that angiotensin II stimulation of quiescent cells in which p27(KIP1) levels are high results in hypertrophy but promotes hyperplasia when levels of p27(KIP1) are low, as in the presence of other growth factors.

Medema et al. (2000) demonstrated that p27(KIP1) is a major target of AFX-like forkhead proteins. They demonstrated that AFX integrates signals from PI3K/PKB (see AKT1; 164730) signaling and RAS (see 190020)/RAL (see 179551) signaling to regulate transcription of p27(KIP1). They demonstrated that p27 -/- cells are significantly less inhibited by AFX activity than their p27 +/+ counterparts.

Dijkers et al. (2002) showed that both cytokine withdrawal and Fkhrl1 (FOXO3A; 602681) activation induced apoptosis in mammalian cell lines through a death receptor-independent pathway. This involved transcriptional upregulation of p27(KIP1) and proapoptotic Bim (BCL2L11; 603827), loss of mitochondrial integrity, cytochrome c release, and caspase activation. PKB protected cells from cytokine withdrawal-induced apoptosis by inhibiting Fkhrl1, resulting in the maintenance of mitochondrial integrity.

Peters and Ostrander (2001) commented on the work of Di Cristofano et al. (2001), demonstrating how cooperation between Cdkn1b and Pten (601728) contribute to suppression of prostate tumors. They gave a useful tabulation of the cytogenetic location of 16 mapped prostate cancer susceptibility loci and candidate genes.

Phosphorylation leads to the ubiquitination and degradation of CDKN1B. Carrano et al. (1999) determined that SKP2 (601436) specifically recognizes phosphorylated CDKN1B predominantly in S phase rather than in G1 phase, and is the ubiquitin-protein ligase necessary for CDKN1B ubiquitination.

Shin et al. (2002) demonstrated a novel mechanism of AKT-mediated regulation of p27(KIP1). Blockade of HER2/NEU (164870) in tumor cells inhibited AKT kinase activity and upregulated nuclear levels of p27(KIP1). Recombinant AKT and AKT precipitated from tumor cells phosphorylated wildtype p27 in vitro. P27 contains an AKT consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) AKT phosphorylated wildtype p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wildtype p27 localized in the cytosol and nucleus, whereas the mutant p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by AKT. Expression of phosphorylated AKT in primary human breast cancers statistically correlated with the expression of p27 in tumor cytosol. Shin et al. (2002) concluded that AKT may contribute to tumor cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

Liang et al. (2002) demonstrated that AKT phosphorylates p27, impairs the nuclear import of p27, and opposes cytokine-mediated G1 arrest. In cells transfected with constitutively active AKT, wildtype p27 mislocalized to the cytoplasm, but mutant p27 was nuclear. In cells with activated AKT, wildtype p27 failed to cause G1 arrest, while the antiproliferative effect of the mutant p27 was not impaired. Cytoplasm p27 was seen in 41% (52 of 128) primary human breast cancers in conjunction with AKT activation and was correlated with a poor patient prognosis. Liang et al. (2002) concluded that their data showed a novel mechanism whereby AKT impairs p27 function that is associated with an aggressive phenotype in human breast cancer.

Viglietto et al. (2002) independently demonstrated that AKT regulates cell proliferation in breast cancer cells by preventing p27(KIP1)-mediated growth arrest. They also showed that threonine at position 157 is an AKT phosphorylation site and causes retention of p27(KIP1) in the cytoplasm, precluding p27(KIP1)-induced G1 arrest.

Gopfert et al. (2003) analyzed fragments of the p27 transcript for their contribution to cell cycle-regulated translation. An element in the p27 5-prime UTR rendered reporter translation cell cycle-sensitive with maximal translation in G1-arrested cells. The 114-bp element contained a G/C-rich hairpin domain that was predicted to form multiple stable stemloops and also overlapped with a small upstream open reading frame (ORF). Both structures contributed to cell cycle-regulated translation. The upstream ORF could be translated in vitro, and its sequence and position were evolutionarily conserved in mouse and chicken. The precise sequence or length of the upstream ORF-encoded peptide were not important for p27 translation, suggesting that ribosomal recruitment to its initiation codon, rather than the translation product itself, contributes to the regulation.

Using NIH-3T3 mouse fibroblasts and mouse embryonic fibroblasts, Kamura et al. (2004) found that Skp2 was the major ubiquitin ligase involved in ubiquitination of nuclear p27(KIP1) at the S and G2 phases, and that a complex made up of Kpc1 (RNF123; 614472) and Kpc2 (UBAC1; 608129) ubiquitinated cytoplasmic p27(KIP1) at G1 phase. Cytoplasmic degradation of p27(KIP1) required p27(KIP1) nuclear export by Crm1 (XPO1; 602559).

The inverse relationship between proliferation and differentiation in osteoblasts has been well documented. Thomas et al. (2004) found that Runx2 (600211), a master regulator of osteoblast differentiation in mammalian cells, was disrupted in 6 of 7 mammalian osteosarcoma cell lines. Immunohistochemical analysis of human osteosarcomas indicated that expression of p27(KIP1) was also lost as tumors lost osteogenic differentiation. Thomas et al. (2004) found that ectopic expression of Runx2 induced growth arrest through p27(KIP1)-induced inhibition of S-phase cyclin complexes, followed by dephosphorylation of RB1 (614041) and G1 cell cycle arrest. They concluded that RUNX2 establishes a terminally differentiated state in osteoblasts through RB1- and p27(KIP1)-dependent mechanisms that are disrupted in osteosarcomas.

White et al. (2006) showed that postmitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, they demonstrated that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate p27(Kip1). White et al. (2006) concluded that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.

Grimmler et al. (2007) found that a conserved tyrosine (Y88) in the CDK-inhibitory domain of human p27 could be phosphorylated by the Src family kinase LYN (165120) and the oncogene product BCR-ABL (see 189980). Phosphorylation of Y88 did not prevent binding of p27 to cyclin A/CDK2, but it caused phosphorylated Y88 and the inhibitory domain of p27 to be ejected from the CDK2 active site, restoring partial CDK activity. This allowed Y88-phosphorylated p27 to be efficiently phosphorylated on thr187 by CDK2, which in turn promoted its SCF-SKP2-dependent degradation.

Chu et al. (2007) showed that the oncogenic kinase SRC (190090) phosphorylated human p27 at Y74 and Y88. SRC inhibitors increased cellular p27 stability, whereas SRC overexpression accelerated p27 proteolysis. SRC-phosphorylated p27 inhibited cyclin E/CDK2 poorly in vitro, and SRC transfection reduced p27/cyclin E/CDK2 complexes. SRC-activated human breast cancer cell lines exhibited reduced p27, and there was a correlation between SRC activation and reduced nuclear p27 in 482 primary human breast cancers. In tamoxifen-resistant breast cancer cell lines, SRC inhibition increased p27 levels and restored tamoxifen sensitivity. Chu et al. (2007) concluded that SRC-mediated phosphorylation of p27 reduces its inhibitory action on cyclin E/CDK2, facilitating subsequent p27 proteolysis.

By yeast 2-hybrid analysis of an adult human heart cDNA library, Hauck et al. (2008) showed that p27 interacted with the C-terminal region of casein kinase-2 (CK2)-alpha-prime (CSNK2A2; 115442). Immunocytochemical analysis of primary rat ventricular cardiomyocytes revealed colocalization of p27 with CK2-alpha-prime. Angiotensin II, a potent inducer of cardiomyocyte hypertrophy, induced proteasomal degradation of p27 in primary rat cardiomyocytes through CK2-alpha-prime-dependent phosphorylation of p27 on ser83 and thr187, which are conserved in humans and rodents. Conversely, unphosphorylated p27 potently inhibited CK2-alpha-prime. Hauck et al. (2008) concluded that downregulation of p27 by CK2-alpha-prime is necessary for development of agonist- and stress-induced cardiac hypertrophy.

MicroRNAs (miRNAs) are short noncoding RNAs that bind to complementary sequences in the 3-prime UTRs of target mRNAs and inhibit their expression. Kedde et al. (2007) showed that the expression of DND1 (609385), an evolutionarily conserved RNA-binding protein, counteracted the inhibitory effect of several miRNAs in human cells and in primordial germ cells of zebrafish by preventing the association of miRNAs with their target mRNAs. Kedde et al. (2007) detailed the effect of DND1 on the downregulation of p27 mRNA by miR221 (MIRN221; 300568) in human cells. Introduction of DND1 abolished the interaction between miR221 and the 3-prime UTR of p27 mRNA and countered the downregulation of p27 expression by miR221. DND1 bound a uridine-rich region in the 3-prime UTR of p27 mRNA that is near the miR221-binding site and prevented miR221 binding. At least 1 of the 2 uridine-rich regions in the p27 3-prime UTR and the RNA-binding domain of DND1 were required to rescue p27 expression.

Cuesta et al. (2009) found that translation of p27 in HeLa cells and HL60 human promyelocytic leukemia cells was cap dependent. Translation via a proposed internal ribosome entry site appeared to be artifactual, resulting from the presence of cryptic promoters in the 5-prime UTR. Cuesta et al. (2009) showed that the dramatic increase in p27 following phorbol ester treatment was not due to increased mRNA levels, but rather to downregulation of MIR181A (see 612742) and relief of MIR181A-dependent translational repression. The 3-prime UTR of p27 mRNA contains 2 possible MIR181A-binding sites, 1 of which overlaps the MIR221-binding site. Both MIR181A-binding sites could repress p27 translation either individually or synergistically.

Lin et al. (2010) showed that although Skp2 inactivation on its own does not induce cellular senescence, aberrant protooncogenic signals as well as inactivation of tumor suppressor genes do trigger a potent, tumor-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic stress-driven senescence neither elicit activation of the p19(Arf) (see 600160)-p53 (191170) pathway nor DNA damage, but instead depend on Aft4 (604064), p27, and p21 (116899). Lin et al. (2010) further demonstrated that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19(Arf)-p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/Pten (601728)-deficient cells and tumor regression in preclinical studies. Lin et al. (2010) concluded that their findings provided proof-of-principle evidence that pharmacologic inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

The HER2-HER3 (ERBB3; 190151) dimer induces cell growth by activating a kinase cascade that includes phosphorylation of p27, resulting in p27 ubiquitination and proteasomal degradation. Trastuzumab blocks the HER2-HER3 interaction and is used to treat breast cancers with HER2 overexpression, although some of these cancers develop trastuzumab resistance. Using small interfering RNA (siRNA) to identify genes involved in trastuzumab resistance, Lee-Hoeflich et al. (2011) identified several kinases and phosphatases that were upregulated in trastuzumab-resistant cancers, including PPM1H (616016). Knockdown of PPM1H by either siRNA or short hairpin RNA induced trastuzumab resistance and increased cell proliferation. Lee-Hoeflich et al. (2011) found that PPM1H protected p27 from degradation by dephosphorylating thr187, thus removing a degradation signal and stabilizing p27-inhibited cell growth.

▼ Mapping
Baens et al. (1995) characterized 117 cDNAs isolated by direct cDNA selection using pools of human chromosome 12p cosmids. Among these, 3 matched previously determined cDNA sequences, including the cyclin-dependent kinase inhibitor referred to as KIP1. STSs were developed for all cosmids. Regional assignment of the STSs by PCR analysis with somatic cell hybrids and fluorescence in situ hybridization (FISH) showed that the loci mapped to 12p13. Martin et al. (1995) mapped this gene, which they referred to as CDKN4, to 12p12.3 by fluorescence in situ hybridization. By PCR-based screening of genomic YAC clones of the CEPH library, they isolated 7 containing the KIP1 gene. In 4 of these YACs, they found a common STS, D12S358, and 1 of the 4 YACs also contained an additional STS, D12S320, which had been located 4 cM apart from D12S358 on the Genethon genetic map. Most of the YACs containing the KIP1 gene had been assigned to chromosome 12 from hybridization data of inter-Alu PCR products from somatic hybrids.

By FISH, Saito et al. (1999) mapped the mouse Kip gene to chromosome 7D3.

▼ Molecular Genetics
Multiple Endocrine Neoplasia, Type IV

In a 48-year-old Caucasian female with primary hyperparathyroidism and a history of pituitary adenoma, consistent with multiple endocrine neoplasia type IV (MEN4; 610755), Pellegata et al. (2006) identified a heterozygous nonsense mutation in the CDKN1B gene (600778.0001). The mutation was also identified in her older sister who had renal angiomyolipoma, her youngest sister, and that sister's teenaged daughter, who reported no symptoms but did not undergo thorough examination. No mutations in the MEN1 gene (613733) were found in the proband or her older sister.

In a Dutch woman with MEN4, Georgitsi et al. (2007) identified a heterozygous truncating mutation in the CDKN1B gene (600778.0002). Tumor tissue from the patient's cervical carcinoma showed loss of heterozygosity for the wildtype allele and negative immunostaining for the p27 protein. The patient was ascertained from a larger cohort of 37 patients, mostly Dutch, who were clinically suspected to have MEN but were negative for mutation in the MEN1 gene. The authors also studied 19 patients with familial acromegaly/pituitary adenomas and 50 Finnish patients with sporadic acromegaly who underwent direct sequencing of the CDKN1B gene; the Dutch woman was the only patient found to carry a CDKN1B mutation.

In a woman with MEN4, Molatore et al. (2010) identified a heterozygous missense mutation in the CDKN1B gene (P69L; 600778.0003). The mutation caused reduced mutant protein levels due to more rapid degradation, had slightly higher cytoplasmic localization compared to wildtype, and lost the ability to bind CDK2. The P69L mutant protein was less effective at suppressing growth of neuroendocrine tumor cells in vitro compared to wildtype. The findings suggested a tumor suppressor role for p27 in neuroendocrine cells. The patient was 1 (3.7%) of 27 individuals with a MEN-like phenotype who was found to carry a CDKN1B mutation.

In a 69-year-old Spanish woman with MEN4, Malanga et al. (2012) identified a heterozygous mutation in the CDKN1B gene (600778.0004). In vitro functional expression studies using a luciferase reporter in HeLa cells showed that the mutation resulted in a significant reduction (30-60%) in transcription and possibly translation. Patient peripheral blood cells showed a significant 3-fold decrease in CDKN1B mRNA levels compared to controls, consistent with haploinsufficiency. The patient was 1 of 15 Spanish individuals with MEN-like features who underwent direct sequencing of the CDKN1B gene and was the only patient found to carry a mutation.

Small Intestine Neuroendocrine Tumors

Using exome- and genome-sequence analysis of small intestine neuroendocrine tumors (SI-NETs), Francis et al. (2013) identified recurrent somatic mutations and deletions in CDKN1B, which encodes p27. Francis et al. (2013) observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NETs.

Other Disease Associations

Chang et al. (2004) analyzed the CDKN1B gene in 188 families with hereditary prostate cancer (see 176807) and found a significant association between the SNP -79C/T (rs34330) and prostate cancer. The -79C allele was overtransmitted from parents to affected offspring, an association that was observed primarily in offspring whose age at diagnosis was less than 65 years. Chang et al. (2004) suggested that germline variants of this gene play a role in prostate cancer susceptibility.

Grey et al. (2013) found biallelic loss of CDKN1B gene expression in a boy with overgrowth and severe neurodevelopmental delay with autism. He also had left-sided strabismus, maldescended testes, and challenging behavior. Array CGH identified a heterozygous, approximately 108-kb deletion on chromosome 12p13 encompassing the 5-prime end of CDKN1B, APOLD1 (612456), and the 5-prime untranslated region of DDX47 (615428). The patient's unaffected mother also carried this deletion. Both individuals had decreased CDKN1B mRNA expression, but only the boy had decreased protein levels. Sanger sequencing showed that the proband also had a de novo heterozygous -73G-A transition in the promoter of the CDKN1B gene that was demonstrated to result in significantly decreased protein expression. Grey et al. (2013) postulated that the neurologic phenotype in the proband fit a recessive model of inheritance and was due to decreased expression of the CDKN1B below a threshold necessary to ensure normal neurodevelopment. The findings were also consistent with a mouse knockout model that has gigantism and hyperplasia of multiple organs (Fero et al., 1996).

▼ Animal Model
Fero et al. (1996) found that targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T-lymphocyte proliferation, and T cells showed enhanced IL2 (147680) responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. That p27 and Rb function in the same regulatory pathway was suggested by the finding that p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of second ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.

Zindy et al. (1999) generated mice with targeted deletions of both the Ink4d (600927) and Kip1 genes. They found that terminally differentiated, postmitotic neurons in these mice reentered the cell cycle, divided, and underwent apoptosis. Zindy et al. (1999) noted that when either Ink4d or Kip1 alone are deleted, the postmitotic state is maintained, suggesting a redundant role for these genes in mature neurons.

Mitsuhashi et al. (2001) described a mouse model in which p27(Kip1) transgene expression was spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts were detectable within 6 hours of doxycycline administration, and maximum nonlethal expression was approached within 12 hours. After 18 to 26 hours of transgene expression, the G1 phase of the cell cycle was estimated to increase from 9 to 13 hours in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus, the data established a direct link between p27(Kip1) and control of G1 phase length in the mammalian central nervous system and unveiled intrinsic mechanisms that constrain the G1 phase length to a putative physiologic maximum despite ongoing p27(Kip1) transgene expression.

Phosphorylation of p27(Kip1) on threonine-187 by CDK2 is thought to initiate the major pathway for p27 proteolysis. To test the importance of this pathway critically in vivo, Malek et al. (2001) replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187. Malek et al. (2001) demonstrated that cells expressing p27 with the T187A change were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Malek et al. (2001) demonstrated a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.

Uchida et al. (2005) generated mice expressing human CDKN1B under the control of the promoter of the rat insulin gene and observed that increased expression of p27 in pancreatic beta cells induced severe diabetes as a result of inhibition of beta-cell proliferation. In mice lacking either insulin receptor substrate-2 (Irs2 -/-; see 600797) or the long form of the leptin receptor (Lepr -/-; see 601007), they found progressive accumulation of p27 in the nucleus of beta cells. Deletion of Cdkn1b ameliorated hyperglycemia in these mouse models of type II diabetes (125853) by increasing islet mass and maintaining compensatory hyperinsulinemia, which the authors attributed predominantly to stimulation of pancreatic beta-cell proliferation. Uchida et al. (2005) concluded that p27 contributes to beta-cell failure in the development of type II diabetes in Irs2 -/- and Lepr -/- (db/db) mice.

Wolfraim and Letterio (2005) found increased numbers of both Cd4 (186940)-positive and Cd8 (see 186910)-positive T cells in p27(Kip1)-deficient mice. However, there was a greater increase in the numbers of Cd8-positive T cells, resulting in a lower Cd4:Cd8 ratio, due in part to enhanced proliferation of naive Cd8-positive T cells, but not naive Cd4-positive T cells, under conditions of limiting Cd28 (186760)-mediated costimulation.

Nmyc (164840) promotes rapid cell division of granule neuron progenitors (GNPs) in mice, and its conditional loss during embryonic cerebellar development results in severe GNP deficiency, perturbs foliation, and leads to reduced cerebellar mass. Since loss of Nmyc triggers precocious expression of Kip1 and Ink4c (CDKN2C; 603369) in the cerebellar primordium, Zindy et al. (2006) disrupted Kip1 and Ink4c in Nmyc-null cerebella and found that this partially rescued GNP cell proliferation and cerebellar foliation. They concluded that expression of NMYC and concomitant downregulation of INK4C and KIP1 contribute to the proper development of the cerebellum.

Sharov et al. (2006) showed that inhibition of BMP (see BMP1, 112264) signaling in mouse keratinocytes altered the development of hair follicles. Microarray and real-time PCR analysis of laser-captured hair matrix cells showed a strong decrease in the expression of p27(Kip1) and increased expression of selected cyclins in the transgenic mice. p27(Kip1) knockout mice showed a similar increase in anagen hair follicles associated with increased cell proliferation in hair bulbs. Alternatively, activation of BMP signaling in human keratinocytes induced growth arrest and stimulated p27(Kip1) expression. Sharov et al. (2006) concluded that p27(Kip1) mediates the effects of BMP signaling on hair follicle size.

Fritz et al. (2002) described a multiple endocrine neoplasia-like autosomal recessive disorder in the rat. Animals exhibiting the mutant phenotype developed multiple neuroendocrine malignancies within the first year of life, including bilateral adrenal pheochromocytoma, multiple extraadrenal pheochromocytoma, bilateral medullary thyroid cell neoplasia, bilateral parathyroid hyperplasia, and pituitary adenoma. The appearance of neoplastic disease was preceded by the development of bilateral juvenile cataracts. Although the spectrum of affected tissues was reminiscent of human forms of MEN, no germline mutations were detected in the RET (164761) or MEN1 (613733) genes. Segregation studies in F1 and F2 crosses yielded frequencies of affected animals consistent with an autosomal recessive mode of inheritance.

In rats with an MEN-like syndrome (Menx), with phenotypic overlap of MEN1 (131100) and MEN2A (171400), Pellegata et al. (2006) performed linkage analysis and identified a locus in a 4-Mb segment on rat chromosome 4, which includes the Cdkn1b gene. Sequencing revealed a homozygous frameshift mutation in the Cdkn1b gene resulting in a dramatic reduction of p27(Kip1) protein.

Besson et al. (2007) generated knockin mice expressing a mutant p27 protein, called p27(CK-), that was unable to interact with cyclins and cyclin-dependent kinases. In contrast to complete deletion of the Cdkn1b gene, which causes spontaneous tumors only in pituitary, p27(CK-) dominantly caused hyperplastic lesions and tumors in multiple organs. The high incidence of spontaneous tumors in lung and retina was associated with amplification of stem/progenitor cell populations.

Karlas et al. (2010) reported the discovery of 287 human host cell genes, including CDKN1B, influencing influenza A virus replication in a genomewide RNA interference screen. Using an independent assay, Karlas et al. (2010) confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. CDKN1B inhibited both viral strains. Furthermore, H1N1 virus-infected p27-null mice accumulated significantly lower viral titers in the lung, providing in vivo evidence for the importance of this gene.

▼ ALLELIC VARIANTS ( 6 Selected Examples):

In a 48-year-old Caucasian female with primary hyperparathyroidism and a history of pituitary adenoma (MEN4; 610755), Pellegata et al. (2006) identified a heterozygous 692G-A transition in the CDKN1B gene, resulting in a trp76-to-ter (W76X) substitution. The mutation was also identified in her older sister who had renal angiomyolipoma, her youngest sister, and that sister's teenaged daughter, who reported no symptoms but did not undergo thorough examination. The mutation was not found in 380 unrelated healthy controls. Molecular and immunohistochemical analysis of the renal angiomyolipoma showed that it retained the CDKN1B wildtype allele and demonstrated RNA expression but showed no p27 protein staining.

Molatore et al. (2010) demonstrated that the mutant W76X protein localized only to the cytoplasm and not to the nucleus. The mutant protein was unable to suppress the growth of neuroendocrine tumor cells in vitro, and lost its pro-apoptotic ability. The truncated protein was expressed in normal kidney tissue from the patient reported by Pellegata et al. (2006), suggesting that it is stable.

In a Dutch woman with multiple endocrine neoplasia type IV (MEN4; 610755), Georgitsi et al. (2007) identified a heterozygous 19-bp duplication in exon 1 of the CDKN1B gene (c.59_77dup19), resulting in a frameshift and premature termination. The patient developed small-cell neuroendocrine cervical carcinoma, an ACTH-secreting pituitary adenoma, and hyperparathyroidism in her forties. She was also diagnosed with multiple sclerosis. Tumor tissue from the cervical carcinoma showed loss of heterozygosity for the wildtype allele and negative immunostaining for the p27 protein.

In a 79-year-old Caucasian woman with multiple endocrine neoplasia type IV (MEN4; 610755), Molatore et al. (2010) identified a heterozygous c.678C-T transition in the CDKN1B gene, resulting in a pro69-to-leu (P69L) substitution. The mutation, which was found by direct sequencing, was not present in the dbSNP database or in 370 control individuals. In vitro cellular expression studies showed that the mutation caused reduced mutant protein levels due to more rapid degradation, as well as slightly higher cytoplasmic localization compared to wildtype. Molecular modeling indicated that the mutation affected a CDK2 (116953)-binding site, and immunoblot analysis confirmed that the mutant protein could not bind CDK2. The P69L mutant protein was less effective at suppressing growth of neuroendocrine tumor cells in vitro compared to wildtype. The patient had bronchial carcinoid, a nonfunctioning pituitary microadenoma, parathyroid adenoma, and papillary thyroid carcinoma. Both bronchial carcinoid and parathyroid adenoma tissue showed decreased or even absent p27 protein expression, but loss of heterozygosity for the wildtype CDKN1B allele was observed only in the carcinoid sample.

In a 69-year-old Spanish woman with multiple endocrine neoplasia type IV (MEN4; 610755), Malanga et al. (2012) identified a heterozygous 4-bp deletion in the 5-prime untranslated region of the CDKN1B gene (c.-32_-29delGAGA). In vitro functional expression studies using a luciferase reporter in HeLa cells showed that the mutation resulted in a significant reduction (30-60%) in transcription and possibly translation. Patient peripheral blood cells showed a significant 3-fold decrease in CDKN1B mRNA levels compared to controls, consistent with haploinsufficiency. The patient had gastric carcinoid tumor and hyperparathyroidism; there was no family history of endocrine neoplasia.

In a 62-year-old woman with multiple endocrine neoplasia type IV (MEN4; 610755), Occhi et al. (2013) identified a heterozygous 4-bp deletion (c.-456_-453delCCTT) in a highly conserved region in the 5-prime untranslated region of the CDKN1B gene. The mutation was not found in the dbSNP or 1000 Genomes Project databases or in 600 control chromosomes. The deletion shifted the upstream open reading frame (ORF) termination codon, thus lengthening the upstream ORF-encoded peptide from 29 to 158 amino acids and shortening the intracistronic space from 429 to 38 bp, with a possible negative influence on translation reinitiation from the main ATG. This change was predicted to prevent proper functioning of the 40S ribosomal subunit during translation. Patient cells showed normal levels of mutant mRNA, but decreased expression of the p27 protein, with weak cytoplasmic staining. Pancreatic tumor cells from the patient showed weak cytoplasmic p27 staining; there was no loss of heterozygosity for the wildtype allele. In vitro functional cellular expression assays showed that the 4-bp deletion impaired translation of a reporter gene by affecting translation reinitiation. The findings elucidated a novel mechanism by which p27 levels can be modulated by changes in the upstream ORF. The patient had acromegaly and a well-differentiated nonfunctioning pancreatic endocrine neoplasm.

In a 53-year-old Italian woman with multiple endocrine neoplasia type IV (MEN4; 610755), Tonelli et al. (2014) identified a heterozygous 2-bp deletion (c.371_372delCT) in exon 1 of the CDKN1B gene, resulting in a frameshift and premature termination at codon 145. The patient had hyperparathyroidism due to parathyroid adenomas and gastrointestinal neuroendocrine tumors; she also had a history of hypothyroidism due to Hashimoto thyroiditis. Analysis of the patient's hyperplastic parathyroid tissue showed reduced nuclear p27 staining, but there was no loss of heterozygosity of the wildtype CDKN1B allele. The patient's asymptomatic 35-year-old son also carried the mutation.

Tags: 12p13.1