p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3 processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.[Keywords: Small nuclear ribonucleoprotein; histone pre-mRNA 3Ј processing; Sm-like protein; Sm core structure; SMN-Gemin complex] Sm and Sm-like (Lsm) proteins have been found in eukaryotes, archaea, and eubacteria. They are characterized by two closely spaced, conserved Sm motifs 1 and 2 (Hermann et al. 1995;Seraphin 1995) which adopt a fold consisting of an ␣-helix followed by five ␤-strands (Kambach et al. 1999b). A common characteristic of Sm/Lsm proteins is their tendency to form oligomers that can close into hepta-or hexameric ring structures which, in turn, control various aspects of RNA metabolism.The seven prototype Sm proteins B/BЈ, D1, D2, D3, E, F, and G form the so-called Sm core structure around the conserved Sm-binding site, RAUU U / G UUGR, of the spliceosomal small nuclear RNAs (snRNAs; Lührmann et al. 1990;Raker et al. 1996;Kambach et al. 1999a;). The formation of this structure occurs in the cytoplasm and was recently shown to be ATP-dependent and mediated by specific assembly factors. A key player in this process is the "survival of motor neurons" (SMN) protein which is mutated in the neuromuscular disorder spinal muscular atrophy (for review, see Paushkin et al. 2002). SMN is part of the so-called SMN complex, which is composed of at least 18 distinct proteins, including all Sm proteins. In vitro reconstitution of the assembly reaction revealed that Sm proteins first associate with the SMN complex and are subsequently transferred to the U snRNA (Meister et al. 2001a;Pellizzoni et al. 2002). The functioning of the SMN complex is regulated by the PRMT5 complex. This complex introduces symmetrical dimethylarginines in Sm proteins B/BЈ, D1, and D3, thereby increasing their affinity for SMN (Brahms et al. 2001;Friesen et al. 2001;Meister et al. 2001b).In contrast to the canonical Sm proteins found in the Sm core of spliceosomal...

The vertebrate heart possesses autoregulatory mechanisms enabling it first to sense and then to adapt its force of contraction to continually changing demands. The molecular components of the cardiac mechanical stretch sensor are mostly unknown but of immense medical importance, since dysfunction of this sensing machinery is suspected to be responsible for a significant proportion of human heart failure. In the hearts of the ethylnitros-urea (ENU)-induced, recessive embryonic lethal zebrafish heart failure mutant main squeeze (msq), we find stretch-responsive genes such as atrial natriuretic factor (anf) and vascular endothelial growth factor (vegf) severely down-regulated. We demonstrate through positional cloning that heart failure in msq mutants is due to a mutation in the integrin-linked kinase (ilk) gene. ILK specifically localizes to costameres and sarcomeric Z-discs. The msq mutation (L308P) reduces ILK kinase activity and disrupts binding of ILK to the Z-disc adaptor protein ␤-parvin (Affixin). Accordingly, in msq mutant embryos, heart failure can be suppressed by expression of ILK, and also of a constitutively active form of Protein Kinase B (PKB), and VEGF. Furthermore, antisense-mediated abrogation of zebrafish ␤-parvin phenocopies the msq phenotype. Thus, we provide evidence that the heart uses the Integrin-ILK-␤-parvin network to sense mechanical stretch and respond with increased expression of ANF and VEGF, the latter of which was recently shown to augment cardiac force by increasing the heart's calcium transients.[Keywords: Integrin-linked kinase (ILK); zebrafish; cardiac stretch sensor; ␤-parvin (Affixin)] Supplemental material is available at http://www.genesdev.org.

Z-disks, the mechanical integration sites of heart and skeletal muscle cells, link anchorage of myofilaments to force reception and processing. The key molecules that enable the Z-disk to persistently withstand the extreme mechanical forces during muscle contraction have not yet been identified. Here we isolated nexilin (encoded by NEXN) as a novel Z-disk protein. Loss of nexilin in zebrafish led to perturbed Z-disk stability and heart failure. To evaluate the role of nexilin in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found several mutations in NEXN associated with the disease. Nexilin mutation carriers showed the same cardiac Z-disk pathology as observed in nexilin-deficient zebrafish. Expression in zebrafish of nexilin proteins encoded by NEXN mutant alleles induced Z-disk damage and heart failure, demonstrating a dominant-negative effect and confirming the disease-causing nature of these mutations. Increasing mechanical strain aggravated Z-disk damage in nexilin-deficient skeletal muscle, implying a unique role of nexilin in protecting Z-disks from mechanical trauma.

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.Several nuclear RNA-protein complexes (RNPs) 3 involved in the processing of mRNAs, such as the snRNPs of the major (U1, U2, U4/6, and U5) and minor (U11, U12, U5, and U4/6atac) spliceosome and the histone-mRNA processing U7 snRNP contain a set of evolutionary conserved proteins of the Sm/LSm class (1, 2). This group of proteins has the propensity to form heptameric rings in the presence of their respective target snRNA. Sm and LSm/Sm rings (also called "cores") can form spontaneously in vitro on their target RNAs (3-5). However, assembly in vivo occurs in a highly regulated manner and is assisted by trans-acting factors. One well characterized entity in this pathway is the SMN complex, whose name-giving component SMN is the product of the spinal muscular atrophy (SMA) disease gene (6, 7). This entity recruits all Sm proteins and promotes their transfer onto the U snRNAs (8 -10). Likewise, assembly of the U7 snRNP is facilitated by a specialized SMN complex that is charged with the unique set of Sm and LSm proteins of this particle (11). With a sedimentation coefficient of 25-40 S and an estimated molecular mass exceeding 1 megadalton, the SMN complex represents a macromolecular machine of great complexity. So far, nine major proteins termed SMN, Gemins 2-8, and unrip as well as nine Sm/LSm protein "substrates" (i.e. B/BЈ, D1, D2, D3, E, F, G, LSm10, and LSm11) have been identified as components of this assembly machinery (12, 13).Our knowledge about the architecture of the core SM...