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At least 19 recordsLinked to original sources

PubMed2005

Binding of HTm4 to cyclin-dependent kinase (Cdk)-associated phosphatase (KAP).Cdk2.cyclin A complex enhances the phosphatase activity of KAP, dissociates cyclin A, and facilitates KAP dephosphorylation of Cdk2.

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex.

Amino Acid Sequence↗

PubMed2003

The L1 major capsid protein of human papillomavirus type 11 interacts with Kap beta2 and Kap beta3 nuclear import receptors.

We have previously shown that the L1 major capsid protein of low-risk HPV11 binds to the Kap alpha2 adapter and enters the nucleus via a Kap alpha2beta1-mediated pathway. In this study, we discovered that HPV11 L1 capsomeres bind to Kap beta2 import receptor, known to mediate nuclear import of hnRNP A1 via interaction with its nuclear localization signal termed M9. Significantly, binding of HPV11 L1 capsomeres to Kap beta2 inhibited the nuclear import of Kap beta2, and its specific M9-containing cargo. Interestingly, HPV11 L1 capsomeres also interacted with Kap beta3 import receptor and inhibited Kap beta3 nuclear import. Moreover, the L1 capsomeres of high-risk HPV-16 shared these activities. These data suggest that HPV L1 major capsid proteins interact with Kap beta2 and Kap beta3, and they may inhibit the Kap beta2- and Kap beta3-mediated nuclear import pathways during the productive phase of the viral life cycle when the virions are assembled and released.

Active Transport, Cell Nucleus↗

PubMed2000

Overexpression of kinase-associated phosphatase (KAP) in breast and prostate cancer and inhibition of the transformed phenotype by antisense KAP expression.

Accumulating evidence suggests that phosphatases play an important role in regulating a variety of signal transduction pathways that have a bearing on cancer. The kinase-associated phosphatase (KAP) is a human dual-specificity protein phosphatase that was identified as a Cdc2- or Cdk2-interacting protein by a yeast two-hybrid screening, yet the biological significance of these interactions remains elusive. We have identified the KAP gene as an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy. Here we report that breast and prostate malignancies are associated with high levels of KAP expression. The sublocalization of KAP is variable. In normal cells, KAP is primarily found in the perinuclear region, but in tumor cells, a significant portion of KAP is found in the cytoplasm. Blocking KAP expression by antisense KAP in a tetracycline-regulatable system results in a reduced population of S-phase cells and reduced Cdk2 kinase activity. Furthermore, lowering KAP expression led to inhibition of the transformed phenotype, with reduced anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings suggest that therapeutic intervention might be aimed at repression of KAP gene overexpression in human breast and prostate cancer.

Animals↗

PubMed2000

Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions.

The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins. KRAB-mediated gene silencing requires binding to the corepressor KAP-1. The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface. Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity. We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components. Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain. A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain. The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions. KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies. Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity. Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding. Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain. The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition. These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins.

Amino Acid Motifs↗

PubMed1992

Effects of pituitary hormones on the cell-specific expression of the KAP gene.

The expression of kidney androgen-regulated protein (KAP) gene in mouse kidney is regulated in a multihormonal fashion. As determined by in situ hybridization analysis, epithelial cells of proximal convoluted tubules of cortical nephrons express KAP mRNA in response to androgenic stimulation while similar cells in the juxtamedullary S3 segment of the tubules express KAP mRNA under estrogenic and pituitary hormonal control. In situ hybridization analysis of kidney sections using hypophysectomized (hypox) mice resulted in a total absence of KAP mRNA suggesting the participation of a pituitary hormone(s) in the constitutive expression of KAP mRNA in S3 cells. Treatment of hypox mice with steroid hormones showed that androgens restored the ability of cortical tubule cells to synthesize KAP mRNA. Estrogen treatment, on the other hand, partially induced KAP gene expression only in S3 cells. These results indicated that the androgenic response of the gene is independent of pituitary function, while expression in S3 cells, although partially induced by the direct action of estrogens, is primarily regulated by a pituitary factor. In order to elucidate which hormone(s) is responsible for KAP gene expression in S3 cells, individual pituitary hormones were administered to hypox normal animals and to strains of mice genetically deficient in certain pituitary hormones. Surgically treated C57BL/6 female and male mice were implanted for 7 days with osmotic pumps containing individual pituitary hormones, after which the kidneys were analyzed by in situ hybridization. Mice injected with growth hormone (GH), corticotropin (ACTH), prolactin (PRL), or vehicle failed to express KAP mRNA. Mice treated with thyrotropin (TSH), follitropin (FSH), and lutropin (LH) exhibited high levels of KAP mRNA in S3 cells of females as well as in the renal cortex of male animals. Expression in the cortex in response to LH and FSH may be due to their gonadotropic effect on testosterone production. Similarly, contamination of TSH samples with small amounts of the gonadotropins may explain the cortical response to TSH. TSH produced the strongest response in S3 cells suggesting that it is responsible for the permissive effect of the pituitary on KAP gene expression. This conclusion was supported by studies performed with the dwarf mouse (dw/dw) which lacks PRL, GH, and TSH due to a mutation in the pit-1 gene. In situ hybridization analysis of dwarf mice kidney sections showed a complete lack of KAP gene expression. The possible participation of GH and PRL was eliminated on the basis of the hormone replacement studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗

PubMed1999

KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing.

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

3T3 Cells↗

PubMed1999

Two novel Krüppel-associated box-containing zinc-finger proteins, KRAZ1 and KRAZ2, repress transcription through functional interaction with the corepressor KAP-1 (TIF1beta/KRIP-1).

We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins.

3T3 Cells↗

PubMed2003

Abolishment of the interaction between cyclin-dependent kinase 2 and Cdk-associated protein phosphatase by a truncated KAP mutant.

The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP) is a human dual-specificity protein phosphatase that dephosphorylates Cdk2 on a conserved threonine residue, T160, in a cyclin dependent manner. Several aberrant KAP transcripts with characteristic deletion regions have been identified in hepatocellular carcinoma tissues. In this report, we demonstrated that multiple aberrant KAP transcripts were also present in a hepatoblastoma cell line (HepG2), albeit harboring a totally different set of deletions. By performing yeast two-hybrid and co-immunoprecipitation experiments, a KAP-Cdk2 interaction domain located in the amino acid 1-34 region was identified. This interaction domain was different from the major protein interface deduced from crystal structure analysis. Using a yeast three-hybrid system, it was shown that the presence of a truncated KAP mutant encoding this interaction domain abolished the wild-type KAP-Cdk2 interaction. In conclusion, a previously unidentified KAP-Cdk2 interaction domain was discovered. Truncated KAP mutants containing this domain interfered with the wild-type KAP-Cdk2 interaction.

Amino Acid Sequence↗

PubMed2007

Aberrant splicing of cyclin-dependent kinase-associated protein phosphatase KAP increases proliferation and migration in glioblastoma.

The cyclin-dependent kinase (Cdk)-associated protein phosphatase KAP is a dual-specificity phosphatase of which the only known function is to dephosphorylate Cdk2 and inhibit cell cycle progression. Paradoxically, we find increased KAP mRNA expression in malignant astrocytomas, which correlates with increasing histologic grade and decreased patient survival. We have resolved this apparent paradox with the discovery of aberrant KAP splicing in malignant astrocytomas that leads to increased expression of KAP-related transcripts but decreased KAP protein expression. In addition, the aberrant splicing generates a dominant negative KAP variant that increases proliferation. We provide the first evidence that KAP not only regulates proliferation but also inhibits migration by decreasing cdc2 mRNA and protein expression. The effect of KAP on cdc2 expression requires its phosphatase activity but does not involve direct dephosphorylation of cdc2. Thus, KAP regulates both cdc2-dependent migration and Cdk2-dependent proliferation, and its loss due to aberrant splicing increases malignancy in human gliomas.

Alternative Splicing↗

PubMed2006

The KRAB-associated co-repressor KAP-1 is a coiled-coil binding partner, substrate and activator of the c-Fes protein tyrosine kinase.

The c-Fes protein tyrosine kinase is implicated in the differentiation of a number of cell types including neuronal, endothelial and myeloid cells. Structurally, Fes consists of a unique N-terminal region, followed by SH2 (Src homology domain 2) and kinase domains. Two coiled-coil (CC) domains (CC1 and CC2) located within the unique N-terminal region are critical regulators of Fes activity in vivo and may function to recruit Fes activators and/or substrates. A yeast two-hybrid screen, utilizing a K-562 cell cDNA library and the Fes CC2 domain as bait, identified an interacting clone encoding the CC domain and B-box motifs (residues 114-357) of the transcriptional co-repressor KRAB-associated protein (KAP)-1. KAP-1(114-357) interacted with full-length Fes in yeast, and the KAP-1 CC domain was sufficient to bind the Fes N-terminal region in Sf-9 cells. Co-expression of Fes with full-length KAP-1 in human 293T cells stimulated Fes autophosphorylation and led to KAP-1 tyrosine phosphorylation. Association of endogenous Fes and KAP-1 was also observed in HL-60 myeloid leukaemia cells. Together, these data identify a novel Fes-KAP-1 interaction, and suggest a dual role for KAP-1 as both a Fes activator and downstream effector.

Animals↗

PubMed2002

Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.

Analysis of the EBI/GeneBank(TM) data base using non-human hair keratin-associated protein (KAP) cDNA sequences as a query resulted in the identification of a first domain of high glycine-tyrosine and high sulfur KAP genes located on human chromosome 21q22.1. This domain, present on the DNA accession numbers and, was approximately 535 kb in size and contained 17 high glycine-tyrosine and 7 high sulfur KAP genes, as well as 9 KAP pseudogenes. Based on amino acid sequence comparisons of the encoded proteins, the KAP genes could be divided into seven high glycine-tyrosine gene families (KAP6-KAP8, and KAP19-KAP22) and four high sulfur gene families (KAP11, KAP13, KAP15, and KAP23). The high glycine-tyrosine genes described here appear to represent the complete set of this type of KAP genes present in the human genome. Both systematic cDNA isolation studies from an arrayed scalp cDNA library and in situ hybridization expression studies of all of the KAP genes identified in the 21q22.1 region revealed varying degrees and regions of expression of 11 members of the high tyrosine-glycine genes and 6 members of the high sulfur KAP genes in the hair forming compartment.

Amino Acid Sequence↗

PubMed2004

Comparative genomics of the keratin-associated protein (KAP) gene clusters in human, chimpanzee, and baboon.

We have previously identified a cluster of 16 genes that encode hair-specific proteins, called keratin-associated proteins (KAPs), located on human Chromosome (Chr) 21q22.3. Here, we have identified similar KAP gene clusters in two primates, chimpanzee and baboon. DNA sequence comparison revealed the common cluster structure consisting of 16 KAP genes for these three primates, but a significant difference was found in the baboon. Baboon possesses a new KAP gene not found in human and chimpanzee, whereas one KAP gene ( KRTAP18.12) that exists in human and chimpanzee was lost in baboon, making no change in the total number of KAP genes. Interestingly, the sequence for coding regions are highly variable among species owing to insertions and deletions, resulting in variation of gene size. On the contrary, the sequences for the 5' upstream region are highly conserved among species. These findings suggest that the ancestral KAP gene cluster was composed of 17 genes before the divergence of Old World monkeys (baboon) to the anthropoid (human and chimpanzee).

Amino Acid Sequence↗

PubMed2004

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos.

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.

Amino Acid Sequence↗

PubMed2004

Hair keratin associated proteins: characterization of a second high sulfur KAP gene domain on human chromosome 21.

Analysis of the EBI/GeneBank database using nonhuman hair keratin associated protein (KAP) gene sequences as a query resulted in the identification of two human KAP gene domains on chromosome 21, one of which, located at 21q22.1, has recently been characterized. The second domain presented here, an approximately 90 kb domain on chromosome 21q23, harbored 16 KAP genes and two KAP pseudogenes. By comparison with known sheep and mouse KAP families, these genes could be assigned to two KAP families, KAP10 and KAP12, with the KAP10 family (12 members) being distinctly larger than the KAP12 family (four members). Systematic cDNA/3' rapid amplification of cDNA ends isolation studies using human scalp mRNA led to the identification of eight KAP10 and two KAP12 cDNA sequences. In situ hybridization analyses of human anagen hair follicles using specific 3'-noncoding sequences of the various KAP10/KAP12 genes revealed mRNA expression of nearly all KAP10 and KAP12 members exclusively in a narrow region of the middle portion of the hair fiber cuticle. Bioinformatic analyses of the promoter regions of the KAP10/KAP12 genes demonstrated several enhancer elements that were present in nearly all of the KAP genes. Primary among these were binding elements for the ETS, heat shock factor, AML, and HOX families of transcription factors.

Amino Acid Sequence↗

PubMed2000

Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: direct chromoshadow domain-KAP-1 corepressor interaction is essential.

The KRAB domain is a highly conserved transcription repression module commonly found in eukaryotic zinc finger proteins. KRAB-mediated repression requires binding to the KAP-1 corepressor, which in turn recruits members of the heterochromatin protein 1 (HP1) family. The HP1 proteins are nonhistone chromosomal proteins, although it is unclear how they are targeted to unique chromosomal domains or promoters. In this report, we have reconstituted and characterized the HP1-KAP-1 interaction using purified proteins and have compared KAP-1 to three other known HP1 binding proteins: SP100, lamin B receptor (LBR), and the p150 subunit from chromatin assembly factor (CAF-1 p150). We show that the chromoshadow domain (CSD) of HP1 is a potent repression domain that binds directly to all four previously described proteins. For KAP-1, we have mapped the CSD interaction region to a 15-amino-acid segment, termed the HP1BD, which is also present in CAF-1 p150 but not SP100 or LBR. The region of KAP-1 harboring the HP1BD binds as a monomer to a dimer of the CSD, as revealed by gel filtration, analytical ultracentrifugation, and optical biosensor analyses. The use of a spectrum of amino acid substitutions in the human HP1alpha CSD revealed a strong correlation between CSD-mediated repression and binding to KAP-1, CAF-1 p150, and SP100 but not LBR. Differences among the HP1 binding partners could also be discerned by fusion to a heterologous DNA binding domain and by the potential to act as dominant negative molecules. Together, these results strongly suggest that KAP-1 is a physiologically relevant target for HP1 function.

3T3 Cells↗

PubMed2002

KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen.

The KAP-2 protein that binds to the H-box (CCTACC) element in the bean CHS15 chalcone synthase promoter was purified, and internal peptide sequence used to design primers leading to the cloning of KAP-2 from bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula). KAP-2 shares sequence similarity to the large subunit of mammalian Ku autoantigen, a protein proposed to be involved in control of DNA recombination and transcription. KAP-2 sequences were present in genomic DNA from a range of legumes, and a related protein is found in Arabidopsis thaliana. Recombinant KAP-2 expressed in insect cells showed the same binding specificity for the CHS15 H-box as the protein purified from bean cell extracts. In vitro transcription assays confirmed that KAP-2 stimulates transcription from a promoter harboring the H-box cis element.

Acyltransferases↗

PubMed2006

Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.

The cellular DNA-damage response is a signaling network that is vigorously activated by cytotoxic DNA lesions, such as double-strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which modulates this process by phosphorylating key players in these pathways. A long-standing question in this field is whether DSB formation affects chromatin condensation. Here, we show that DSB formation is followed by ATM-dependent chromatin relaxation. ATM's effector in this pathway is the protein KRAB-associated protein (KAP-1, also known as TIF1beta, KRIP-1 or TRIM28), previously known as a corepressor of gene transcription. In response to DSB induction, KAP-1 is phosphorylated in an ATM-dependent manner on Ser 824. KAP-1 is phosphorylated exclusively at the damage sites, from which phosphorylated KAP-1 spreads rapidly throughout the chromatin. Ablation of the phosphorylation site of KAP-1 leads to loss of DSB-induced chromatin decondensation and renders the cells hypersensitive to DSB-inducing agents. Knocking down KAP-1, or mimicking a constitutive phosphorylation of this protein, leads to constitutive chromatin relaxation. These results suggest that chromatin relaxation is a fundamental pathway in the DNA-damage response and identify its primary mediators.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2003

Targeting of alpha-kinase-anchoring protein (alpha KAP) to sarcoplasmic reticulum and nuclei of skeletal muscle.

The sarcoplasmic reticulum (SR) plays a key role in excitation/contraction coupling of skeletal muscle. The SR is composed of two continuous yet heterogeneous membrane compartments, the free or longitudinal SR and cisternal SR. Cisternal SR is made up of free SR membrane, enriched in Ca(2+) pumps, and junctional SR (jSR) membrane, enriched in ryanodine-sensitive Ca(2+)-release channels, and contains calsequestrin within its lumen. Protein phosphorylation mediated by the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) has significant, distinct regulatory roles in both Ca(2+) uptake and Ca(2+) release. Kinase-anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of alpha KAP, a CaM kinase II-anchoring protein encoded within the alpha-CaM kinase II gene, was studied in transgenic skeletal muscle fibres of the adult rat soleus. The transgenes were epitope-tagged versions of alpha KAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that alpha KAP is largely localized at the free SR and thus near the Ca(2+) pump, a protein that can be modulated by CaM kinase II phosphorylation. Only minor co-localization was observed with the jSR ryanodine-sensitive Ca(2+)-release channel, which is a potential CaM kinase II target. In non-muscle cells, recombinant alpha KAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of alpha KAP. An unexpected additional specific localization that does not require the N-terminus was found in the nucleus, providing a first clue of how CaM kinase II can fulfil its nuclear functions in skeletal muscle.

Amino Acid Sequence↗