Protein phosphatase 2A is a heterotrimeric protein serine/threonine phosphatase consisting of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. The B subunits determine the substrate specificity of the enzyme. There have been three families of cellular B subunits identified to date: B55, B56 (B'), and PR72/130. We have now cloned five genes encoding human B56 isoforms. Polypeptides encoded by all but one splice variant (B56gamma1) are phosphoproteins, as shown by mobility shift after treatment with alkaline phosphatase and metabolic labeling with [32P]phosphate. All labeled isoforms contain solely phosphoserine. Indirect immunofluorescence microscopy demonstrates distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta, and B56epsilon complexed with the protein phosphatase 2A A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1, and B56gamma3 are concentrated in the nucleus. Two isoforms (B56beta and B56delta) are highly expressed in adult brain; here we show that mRNA for these isoforms increases severalfold when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment. These studies demonstrate an increasing diversity of regulatory mechanisms to control the activity of this key intracellular protein phosphatase and suggest distinct functions for isoforms targeted to different intracellular locations.

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I (CKI) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKI binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKI leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKI may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.

Casein kinase I ⑀ (CKI⑀) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKI⑀ activity and substrate specificity are not well understood. We report that CKI⑀, which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKI␣, is substantially less active than CKI␣ in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKI⑀ have been identified. First, limited tryptic digestion of CKI⑀ produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKI⑀ activates CKI⑀ 5-20-fold toward T antigen. Similar treatment of a truncated form of CKI⑀ produced only a 2-fold activation. Notably, this activation was transient; reautophosphorylation led to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activated CKI⑀ toward the novel substrates IB␣ and Ets-1. These mechanisms may serve to regulate CKI⑀ and related forms of CKI in the cell, perhaps in response to DNA damage. The casein kinase I (CKI)1 gene family encompasses an increasing number of genes expressed in eukaryotes including yeast Caenorhabditis elegans and mammals. Two subgroups of the CKI family have been separated by functional analysis and complementation of mutations in yeast. One group encoding nuclear kinases appears in yeast to be involved in the response to DNA damage. Mutations in these genes, including HRR25 and YCK3 in Saccharomyces cerevisiae and hhp1 and hhp2 in Schizosaccharomyces pombe, lead to sensitivity to DNA damaging agents such as x-rays and methyl methanesulfonate (1-4). The mammalian genes encoding CKI␦ and CKI⑀ complement HRR25-deleted yeast, suggesting they too may be involved in DNA repair pathways (5). A second group in S. cerevisiae encodes prenylated membrane-bound isoforms involved in bud growth and includes YCK1 and YCK2 (6); deletions in these genes are complemented by the mammalian genes encoding CKI␥ (7).The structure of the CKI family suggests several potential mechanisms for the regulation of activity. All family members contain a short amino-terminal domain of 9 -76 amino acids, a highly conserved kinase domain of 284 amino acids, and a highly variable carboxyl-terminal domain that ranges from 24 to more than 200 amino acids in length. The carboxyl terminus of a CKI isoform may serve several functions, including regulation of substrate recognition, modulation of catalytic activity, and/or determination of kinase subcellular localization. Prenylation of the tail of the YCK1/YCK2 family has been shown to be of functional importance in yeast (3,8,9). Phosphorylation of CKI may also be an important regulatory mechanism. Most of the CKI proteins are phosphoproteins, and several of the yeast kinases can autophosphorylate on serine, threonine, and tyrosine residue...

We have identified a novel glycoprotein expressed exclusively in frog olfactory neuroepithelium, which we have named "olfactomedin". Olfactomedin is a 57-kDa glycoprotein recognized by seven monoclonal antibodies, previously shown to react solely with proteins of olfactory cilia preparations. It undergoes posttranslational modifications, including dimerization via intermolecular disulfides and attachment of complex carbohydrate moieties that contain N-acetylglucosamine and beta-D-galactoside sugars. Olfactomedin strongly binds to Ricinus communis agglutinin I and has been purified to homogeneity by lectin affinity chromatography. Polyclonal rabbit antiserum raised against purified olfactomedin confirmed that it is expressed only in olfactory tissue. Immunohistochemical studies at the light microscopic and electron microscopic level show that olfactomedin is localized in secretory granules of sustentacular cells, in acinar cells of olfactory glands, and at the mucociliary surface. The massive production of olfactomedin and its striking deposition at the chemosensory surface of the olfactory neuroepithelium suggest a role for this protein in chemoreception.

Casein kinase I ␦ (CKI␦) and casein kinase I ⑀ (CKI⑀) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxylterminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.The casein kinase I (CKI) 1 gene family contains two major subgroups that regulate cytoplasmic and nuclear processes, respectively. The nuclear family appears involved in the response to DNA damage in mammals and yeast, whereas the cytoplasmic members are involved in membrane structure and bud morphogenesis (1-5). The founding member of the nuclear family, HRR25, was first cloned in a screen for budding yeast mutants sensitive to DNA double strand breaks (3). In Schizosaccharomyces pombe, the essential gene pair hhp1 ϩ and hhp2 ϩ performs similar functions (4). The mammalian homologs of these genes are the closely related CKI⑀ and CKI␦; they encode monomeric protein kinases that are 97% identical to each other over the kinase domain and 53% identical over their 124 amino acid carboxyl-terminal extensions (6, 7). In fact, the human CKI⑀ gene partially complements Saccharomyces cerevisiae with a deletion of the HRR25 gene (7). A number of the substrates identified to date for the CKI family also support a role for the kinase in regulation of DNA repair and replication. For example, phosphorylation of SV40 large T antigen blocks its ability to unwind the SV40 origin of replication (8 -11). CKI also phosphorylates several isoforms of the rad24 and rad25-related 14 -3-3 proteins, regulating their ability to bind to substrate proteins (12). Purified CKI⑀ can phosphorylate the carboxyl terminus of IB␣, potentially regulating its degradation rate (10, 13). Hrr25 protein binds to and phosphorylates the yeast transcription factor Swi6 (14). Most recently, CKI⑀ and CKI␦ have been identified as the cellular kinases that constitutively phosphorylate the extreme amino terminus of p53 (5).CKI genes appear widely and constitutively expressed, but despite the progress in defining a role for CKI isoforms in the cell, a detailed understanding of the regulation of CKI activity is lacking. Several lines of evidence suggest the carboxyl-terminal tails of CKI␦ and CKI⑀ contain an important autoregulatory domain. First, CKI⑀, with a 124-amino acid carboxylterminal exte...