Role of p21(WAF1/CIP1) as an attenuator of both proliferative and drug-induced apoptotic signals in BCR-ABL-transformed hematopoietic cells.

The constitutive tyrosine kinase activity of the BCR-ABL fusion protein plays a crucial role in the pathogenesis of chronic myeloid leukemia and promotes growth factor-independent survival of hematopoietic cells. In 32D cells, expression levels of retrovirally transduced BCR-ABL were positively correlated with the levels of the cell cycle regulator protein p21, and this upregulation of p21 expression depended on the kinase activity of BCR-ABL. To assess the role of p21 on BCR-ABL-positive hematopoietic cells, we compared proliferation and drug-induced apoptosis in bone marrow (BM) cells from wild-type and p21 knockout mice after retroviral transfer of the BCR-ABL fusion gene. As compared with wild-type cells, p21 knockout cells showed increased proliferation, suggesting that p21 acted as an attenuator of BCR-ABL-mediated cell proliferation. In marked contrast, deletion of p21 promoted apoptosis induction by imatinib and taxol in BCR-ABL-transformed BM cells. These findings demonstrate that p21 has a dual function in BCR-ABL-transformed murine BM cells: It attenuates the effects of two apparently opposed phenomena such as BCR-ABL-mediated cell proliferation and drug-induced apoptosis. This dual function of p21 calls for a cautious evaluation of the suitability of p21 as a secondary target in anticancer therapy.

Src family tyrosine kinases phosphorylate Flt3 on juxtamembrane tyrosines and interfere with receptor maturation in a kinase-dependent manner.

The receptor tyrosine kinase (RTK) Flt3 is expressed in early hematopoietic progenitor cells and stimulates their growth. Due to frequent mutations in the Flt3 gene in patients with acute myeloid leukemia (AML), Flt3 is regarded as a potential therapeutic target, but the underlying mechanisms are still poorly understood. Therefore, we investigated interactions of Flt3 and some Src family tyrosine kinases (SFKs), which are expressed predominantly or exclusively in hematopoietic cells and known to be involved in signal transduction by various RTKs. Employing sets of wt and mutant Flt3 and Hck, we analyzed protein binding as well as Flt3 phosphorylation and maturation in HEK-293 cells cotransfected with expression constructs encoding both binding partners. Kinase-inactive Hck-K269R was recruited to phosphotyrosine residues located in the juxtamembrane (JM) region of activated Flt3 via its SH2 domain. Several of the JM domain tyrosines were phophorylated by Hck and other SFKs. As apparent from the distribution of mature and hypoglycosylated Flt3, SFKs interfered with Flt3 maturation in a kinase-dependent manner. Together, these findings show a complex role of SFKs in Flt3 signaling and reveal a new function of SFKs in the maturation of RTKs.

An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice.

An orally bioavailable and blood-brain barrier penetrating analog of the kinase inhibitor K252a was able to prevent the typical motor deficits in the tau (P301L) transgenic mouse model (JNPL3) and markedly reduce soluble aggregated hyperphosphorylated tau. However, neurofibrillary tangle counts were not reduced in the successfully treated cohort, suggesting that the main cytotoxic effects of tau are not exerted by neurofibrillary tangles but by lower molecular mass aggregates of tau. Our findings strongly suggest that abnormal tau hyperphosphorylation plays a critical role in the development of tauopathy and suggest a previously undescribed treatment strategy for neurodegenerative diseases involving tau pathology.

Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells.

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF-induced response, leading to differentiation of almost all cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.