Proteins that interact with 14-3-3 isoforms are involved in regulation of the cell cycle, intracellular trafficking/targeting, signal transduction, cytoskeletal structure and transcription. Recent novel roles for 14-3-3 isoforms include nuclear trafficking the direct interaction with cruciform DNA and with a number of receptors, small G-proteins and their regulators. Recent findings also show that the mechanism of interaction is also more complex than the initial finding of the novel phosphoserine/threonine motif. Non-phosphorylated binding motifs that can also be of high affinity may show a more isoform-dependent interaction and binding of a protein through two distinct binding motifs to a dimeric 14-3-3 may also be essential for full interaction. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions. In many cases, they show a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of dimer formation may influence which of the 14-3-3-interacting proteins could be brought together. Mammalian and yeast 14-3-3 isoforms show a preference for dimerization with specific partners in vivo.

Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKI␣ co-purifies with centaurin-␣ 1 in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-␣ 1 associates in vitro with all mammalian CKI isoforms. To date, CKI␣ represents the first protein partner identified for centaurin-␣ 1 . However, our data suggest that centaurin-␣ 1 is not a substrate for CKI␣ and has no effect on CKI␣ activity. Centaurin-␣ 1 has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-␣ 1 contains a cysteine-rich domain that is shared by members of a newly identified family of ADPribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKI␣ in these biological events.

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Casein kinase IK K (CKIK K) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIK K interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin-K K 1 , synaptotagmin IX and CPI-17 that were isolated from brain as CKIK K co-purifying proteins. Actin, importin-K K 1 , importin-L L, PP2Ac, centaurin-K K 1 , and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214^233 of CKIK K. We have previously shown that centaurin-K K 1 represents a CKIK K partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIK K and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217^233 of CKIK K which are proposed from X-ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIK K complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIK K protein partners and their relations to CKI are discussed. ß

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Calcitonin gene-related peptide (CGRP) is a 37 amino acid vasodilatory peptide, of which two isoforms, a -C G R P and p-CGRP are known. The use of C-terminal fragments of CGRP, such as C G R P (8-37)Jcd to a pharmacological subdivision of C G R P receptors into CGRP-1 and CGRP-2. We have recently developed the non-peptidc C G R P antagonist, BIBN4096BS, which has sub-nanomolar affinity for primate C G R P receptors. Furthermore, BlBN4096BS is a potent blocker (pKB >lo) of CGRP-induced CAMP stimulation in SK-N-M C cells, human brain microvascular and astroglial cells, CGRP-induced vasodilatation in human temporal arteries, and blocks ncurogenic vasodilatation in the marmoset. BIBN4096 also discriminates between the actions of a -C G R P and [Cys(Et)2,7]hCGRPa in some rat tissues, providing evidence for additional functional C G R P receptor heterogeneity.To further exploit BlBN4096BS as a pharmacological tool, we used BIBN4096BS in porcine coronary vcsscls and cerebral basilar arteries and compared functional with molecular data on C G R P receptor componcnts.The pKB values far BIBN4096BS against [Cys(Et)2,7]hCGRPa, and a -C G R P did not differ significantly (pkB-8), whilst vasodilation of p-CGRP was blocked less potently (pKB-7).Thc analysis of molecular components of the C G R P receptor demonstrated the presence o f hRAMPl and 2, hCRLR and hRCP mRNA suggesting the presence of C G R P l and ADM receptors. O u r data suggest that apart from a isubdivisioni in CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of a -C G R P and p-CGRP. However, evidence for C G R P receptor heterogeneity on a molecular lcvcl is scarce.RAMPs arc proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) o r CTR-like receptor (CRLR). I n this study we screened a range of class I1 G protein-coupled receptors (PTHI, PTH2, G H R H , VPAC1, VPACZ receptors) for possible RAMP interactions by measurement of receptor-induced translocation of epitope-tagged RAMPs. Of these, only the VPACl receptor caused significant translocation of all 3 RAMPs, however the PTH2 receptor also translocated RAMP3 to the cell surface. Unlike CTR and CRLR, co-transfcction of VPACl and RAMPs did not alter 1251-VIP (vasoactive intestinal polypeptide) binding and specificity. Furthermore, in contrast to CTR-RAMP interaction, where there was an incrcase in CAMP accumulation and a decrease in phosphoinositol hydrolysis, there was a specific increase in agonist-mediated phosphoinositol hydrolysis when the VPACl receptors were cotransfected with RAMP2. This change was due to an enhancement of Emax with no change in the EC50 value for VIP. N o significant change in CAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with G protein-coupled receptors outside the C T R family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signalling.The sequencing of human genome is essentially complete and we know its complement of ...