Passive mechanical properties and related proteins change with botulinum neurotoxin A injection of normal skeletal muscle.

The effects of botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Neurotoxin-treated single fiber passive elastic modulus was reduced compared to control fibers (53.00 kPa vs. 63.43 kPa). Fiber stiffness and slack sarcomere length were also reduced compared to control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77% after treatment. Fiber bundle passive elastic modulus increased following treatment (168.83 kPa vs. 75.14 kPa). Bundle stiffness also increased while collagen content per mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal muscle that is opposite to the changes observed in spastic muscles.

The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis.

The intrinsic damage response is activated by DNA damage that arises during the cell division process. The ability of the cell to repair this damage during proliferation is important for normal cell growth and, when disrupted, may lead to increased mutagenesis and tumorigenesis. The atypical CDK activator, Spy1, was previously shown to promote cell survival, prevent apoptosis and inhibit checkpoint activation in response to DNA damage. Prior studies have shown that Spy1 is upregulated in breast carcinomas and accelerates mammary tumorigenesis in vivo. In this report, first, we demonstrate that the ability of Spy1 to inhibit apoptosis and bypass UV-induced checkpoint activation is dependent on the presence of the gene regulatory protein p53 and the CKI p21. Second, we demonstrate that Spy1 expression has the following effects: prevents repair of cyclobutane pyrimidine dimers through bypass of nucleotide excision repair; increases the cellular mutation frequency; and reduces the formation of cyclin E induced gammaH2A.X foci. Lastly, we show that knockdown of endogenous Spy1 leads to gammaH2A.X foci formation, Chk1 phosphorylation and proliferation defects, demonstrating a functional role for Spy1 in the intrinsic DNA damage response. These results also demonstrate that Spy1 fulfills a novel regulatory role in the intrinsic DNA damage response and maintains the balance between checkpoint activation, apoptosis, repair and cell cycle progression in response to exogenous or intrinsic damage. Furthermore, the overexpression of Spy1 as a contributing factor in cancer progression will most likely be confined to p53-positive cells.

Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27.

The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G1 and throughout S-phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S-phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G1 and throughout S-phase.

Speedy/RINGO regulation of CDKs in cell cycle, checkpoint activation and apoptosis.

Speedy/RINGO family members bind and activate cyclin dependent kinases (CDKs), although these proteins have no homology to known cyclin proteins. Members of this family are required for and enhance meiotic maturation, in addition to having novel roles in regulating the mitotic mammalian cell cycle and the DNA damage response. Here we discuss how the specialized functions of these proteins differ from classical cyclin-mediated activation of CDKs. Through atypical activation of CDKs, bypass of conventional inhibitory mechanisms, and unique substrate selection, Speedy/RINGO proteins contribute to cell cycle, checkpoint, and apoptotic regulation. Furthermore, we address the recently established correlation between Spy1 and cancer in terms of the specialized functions of the Speedy/RINGO family.

Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation.

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation.

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.