Abelson phosphorylation of CLASP2 modulates its association with microtubules and actin.

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton.

Information management to enable personalized medicine: stakeholder roles in building clinical decision support.

BACKGROUND: Advances in technology and the scientific understanding of disease processes are presenting new opportunities to improve health through individualized approaches to patient management referred to as personalized medicine. Future health care strategies that deploy genomic technologies and molecular therapies will bring opportunities to prevent, predict, and pre-empt disease processes but will be dependent on knowledge management capabilities for health care providers that are not currently available. A key cornerstone to the potential application of this knowledge will be effective use of electronic health records. In particular, appropriate clinical use of genomic test results and molecularly-targeted therapies present important challenges in patient management that can be effectively addressed using electronic clinical decision support technologies. DISCUSSION: Approaches to shaping future health information needs for personalized medicine were undertaken by a work group of the American Health Information Community. A needs assessment for clinical decision support in electronic health record systems to support personalized medical practices was conducted to guide health future development activities. Further, a suggested action plan was developed for government, researchers and research institutions, developers of electronic information tools (including clinical guidelines, and quality measures), and standards development organizations to meet the needs for personalized approaches to medical practice. In this article, we focus these activities on stakeholder organizations as an operational framework to help identify and coordinate needs and opportunities for clinical decision support tools to enable personalized medicine. SUMMARY: This perspective addresses conceptual approaches that can be undertaken to develop and apply clinical decision support in electronic health record systems to achieve personalized medical care. In addition, to represent meaningful benefits to personalized decision-making, a comparison of current and future applications of clinical decision support to enable individualized medical treatment plans is presented. If clinical decision support tools are to impact outcomes in a clear and positive manner, their development and deployment must therefore consider the needs of the providers, including specific practice needs, information workflow, and practice environment.

Use of a chemical genetic technique to identify myosin IIb as a substrate of the Abl-related gene (Arg) tyrosine kinase.

Abl family kinases have been implicated in the regulation of cell morphogenesis and migration, but the molecular mechanisms through which they operate are not fully elucidated. We applied the bump-hole technique, pioneered by Shokat and colleagues, to identify direct substrates of Abl and the Abl-related gene (Arg) kinases. This technique required the engineering of Abl/Arg to utilize an unnatural ATP analogue as a phospho-donor. Mutation of T334A and T361A in Abl and Arg, respectively, altered their nucleotide specificity and allowed them to utilize N6-benzyl-ATP as a phospho-donor. These mutations did not affect the catalytic activity or protein substrate specificity of Abl and Arg. An unexpected high level of background labeling necessitated further optimization of this approach. Dialysis, pretreatment with a broad-spectrum Ser/Thr kinase inhibitor, K-252a, and purification of phosphotyrosine-containing proteins allowed for definitive identification of putative substrates. Using mass spectrometry, we identified eight putative substrates. One of these putative substrates, myosin IIB, can be phosphorylated in vivo by Arg. Our results indicate that the bump-hole technique can be used to identify Abl family kinase substrates and suggests that myosin IIB may be regulated by tyrosine phosphorylation.

Dissecting kinase signaling pathways.

Aberrant protein kinase signaling is a hallmark of many human diseases including cancer, diabetes, and neurological disorders. Kinase inhibitors have shown to be successful at treating some of these diseases, implying that understanding kinase signaling pathways may lead to additional, non-kinase drug targets. However, identifying substrates of protein kinases is difficult due to the universality of the chemical mechanism kinases utilize and the ability of multiple kinases to phosphorylate the same protein substrates. In this review, we explore the advantages and disadvantages of several techniques for identifying kinase substrates. Once putative substrates are identified, their validation as physiological substrates remains a major challenge. We propose three criteria for confirming the physiological relevance of a putative substrate's interaction with a kinase.

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation.

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.