RESUMO
The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Cinética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfatidilinositol 4,5-Difosfato/metabolismo , Mutação Puntual , Profilinas , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes.
Assuntos
Oligodendroglia/citologia , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/citologia , Regulação para Cima , Animais , Encéfalo/citologia , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Proteínas da Mielina/análise , Proteínas da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/enzimologia , Oligodendroglia/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fyn , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Células-Tronco/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoAssuntos
Biologia Molecular/métodos , Miristatos/metabolismo , Palmitatos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Acilação , Animais , Proteína Tirosina Quinase CSK , Ácidos Graxos/metabolismo , Radioisótopos do Iodo/química , Marcação por Isótopo , Camundongos , Miristatos/química , Palmitatos/química , Rádio , Trítio/química , Domínios de Homologia de src , Quinases da Família srcRESUMO
A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.
Assuntos
Vírus do Sarcoma Aviário/química , Proteínas dos Retroviridae/química , Proteínas da Matriz Viral/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Conformação Proteica , Proteínas dos Retroviridae/genética , Alinhamento de Sequência , Análise de Sequência , Relação Estrutura-Atividade , Proteínas da Matriz Viral/genéticaRESUMO
Members of the Src family of protein tyrosine kinases are localized to subspecialized regions of the plasma membrane. Herein we show that the N-terminal SH4 region of the Src family member p59fyn (Fyn) is both necessary and sufficient for targeting of Fyn and heterologous proteins to the plasma membrane and detergent-insoluble subdomains. Attachment of the first 16 amino acids of Fyn to a normally cytosolic protein, beta-galactosidase, resulted in distinct plasma membrane localization of the chimeric protein. Mutation of the palmitoylation site (cysteine-3) within Fyn16-beta-galactosidase or wild-type Fyn abrogated plasma membrane localization, resulting in redistribution of the mutant proteins into intracellular membranes. Substitution of the SH4 motif within Fyn with heterologous sequences from other palmitoylated proteins (G alpha o and GAP43) revealed that the presence of palmitate is sufficient to direct plasma membrane localization independent of surrounding amino acid sequences and myristate. Palmitoylated Fyn chimeras were also enriched in the Triton X-100-resistant matrix, whereas nonpalmitoylated forms of these proteins were detected in the detergent-soluble fraction. The palmitate moiety on Fyn exhibited a half-life of 1.5-2 h. In contrast, the half-life of the polypeptide backbone was 8 h, indicating that palmitoylation is a reversible modification. These studies establish that the palmitoylated SH4 sequence of Fyn can be used to specifically target proteins to the plasma membrane in a reversible manner.
Assuntos
Membrana Celular/metabolismo , Palmitatos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Compartimento Celular , Camundongos , Miristatos/metabolismo , Octoxinol , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes de Fusão , Proteínas RecombinantesRESUMO
Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases.
Assuntos
Linfócitos B/metabolismo , Ácidos Palmíticos/metabolismo , Quinases da Família src/química , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Linfócitos B/imunologia , Antígenos CD79 , Células COS , Mutagênese Sítio-Dirigida , Ácidos Palmíticos/química , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Especificidade por Substrato , Transfecção , Quinases da Família src/genéticaRESUMO
Nerve growth factor (NGF) represents a family of structurally related trophic factors, including brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5. These neurotrophin factors interact with two classes of receptors, the trk receptor tyrosine kinase family, and the low affinity p75 neurotrophin receptor. To study potential ligand-receptor interactions, recombinant trk fusion proteins have been constructed, and pan-trk polyclonal antisera directed against the cytoplasmic tyrosine kinase domain have been generated. The recombinant proteins were assessed for in vitro kinase activity and for the ability of K-252a to inhibit phosphorylation. Antibodies made against the fusion protein recognize all trk family members, and are effective in immunoprecipitation of affinity-crosslinked receptors. Comparative crosslinking indicates that NGF can recognize all trk receptor members, illustrating the large number of potential ligand-receptor interactions between neurotrophins and their receptors.