A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ~200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and the dimerization of Grb2, which provides the crosslinking element for condensation with EGFR. The Grb2 Y160 residue plays a structurally critical role in dimer formation, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that EGFR condensation controls the ability of SOS to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5null/null mast cells and Stat5ΔN T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5null/null fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.

Studies on the Retention of CF1 with or without Induced ATPase Activity by Pyrophosphate Treated Thylakoids and Its Relation to the Regeneration of Photophosphorylation

The reconstitution of chloroplast coupling factor 1 (CF1) into thylakoid membranes was investigated by the fluorescence of the covalently attached label fluorescamine. In contrast to a func­tional regeneration of ATP synthesis, a rebinding of CF1 was observed regardless if the protein was in its native, purified state or had been activated for ATPase activity by heat, dithiothreitol (DTT) or trypsin treatment. The reintegration of CF1 into the thylakoid membrane was estimated to be almost quantitative. Since the label binds covalently and irreversibly to free NH2-groups, its effects on the various functional parameters were investigated. Blocking of NH2 groups leads to inhibition of ATPase activity in isolated CF1 . Labeled factor used in a reconstitution experiment on the other hand, does still result in considerable regeneration of ATP formation. Attaching the label to whole thylakoids leads to a differential effect on phosphorylation, which is inhibited, and the H+-uptake which seems largely unaffected. A differential effect is also observed on coupled and uncoupled electron transport. The results are interpreted as evidence for an involvement of free NH2-groups in the mechanism of photophosphorylation.