SUMMARY Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprise a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

The Rous sarcoma virus oncogene product, pp60v-src, transforms cultured fibroblasts but its corresponding proto-oncogene product, pp60c-src, does not. Both proteins are known to be protein-tyrosine kinases. Published results suggest that the kinase activity of pp60c-src is inhibited relative to that of pp60v-src, due perhaps to phosphorylation of a tyrosine in pp60c-src that is not phosphorylated in pp60v-src. In this study, it was observed that the tyrosine phosphorylated in pp60c-src is Tyr527, six residues from the COOH-terminus of the protein. The region of pp60c-src from residue 515 to the COOH-terminus, including Tyr527, has been replaced with a different sequence in pp60v-src. Thus, the increase in transforming ability and kinase activity that occurred in the genesis of pp60v-src may have resulted from the loss of a tyrosine involved in negative regulation.

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To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the ␤ subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 M) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G 0 /G 1 than do wild-type cells. Prolongation of G 0 /G 1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.The cellular gene c-src and its viral homolog v-src (the transforming gene of Rous sarcoma virus) encode 60-kDa, cytoplasmic, membrane-associated protein-tyrosine kinases (reviewed in reference 6). For the viral protein (v-Src) or for transforming mutants of the cellular protein (c-Src or Src), a close correlation exists between elevated specific kinase activity and cell transformation. At least four factors are known to influence Src activity: (i) mutation within the coding region of the src gene, (ii) phosphorylation on Tyr 527 and Tyr 416 of Src, (iii) subcellular localization of Src and its substrates, and (iv) association of Src with other cellular proteins.Compelling evidence indicates that Src-binding proteins can regulate Src activity (reviewed in reference 6). A number of interacting proteins that upregulate Src activity have been identified. The prototype is middle T antigen (mT), the transforming protein of polyomavirus. Src complexed with mT has elevated specific activity due to dephosphorylation of Tyr 527 (5,8,11,16). Activation of Src is required for the induction of mammary tumors in mT transgenic mice (24). Characterization of the Src-mT complex led to discovery of the fundamental mechanism by which the cellular Src protein is converted to a transforming protein and defined the requirement of Src for polyomavirus transformation. Thus, characterization of a single Src-binding protein contributed substantially to our understanding of both RNA and DNA tumor virology.While a number of interacting proteins that upregulate Src activity have been identified, few that downregulate Src activity have been identifi...

RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src proteintyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells.The Src family of intracellular protein-tyrosine kinases participates in diverse signaling pathways that regulate cell growth, differentiation, adhesion, and architecture (reviewed in Ref. 1). Identification of Src-binding proteins has led to better understanding of Src regulation and has provided clues about the function of Src in normal and transformed cells. For example, characterization of the interaction between Src and polyoma middle T antigen led to discovery of a fundamental mechanism by which the cellular Src protein is converted to a transforming protein (by dephosphorylation at Tyr-527) and defined the requirement of Src for polyoma transformation (2-7). Thus, characterization of a single Src-binding protein contributed substantially to our understanding of both RNA and DNA tumor biology.Recently, using the unique domain/SH2/SH3 domain of Src as bait, and a human lung fibroblast cDNA library as prey, we identified RACK1, a known intracellular receptor for activated C kinase (RACK), 1 as a Src-binding protein (8). We found that overexpression of RACK1 inhibited the specific activity of Src tyrosine kinases (as measured in vitro) and the growth of NIH 3T3 cells. RACK1 exerted its effect on growth, in part, by prolonging the G 0 /G 1 phase of the cell cycle. RACK1 was the first of a group of proteins (collectively called RACKs) to be identified and characterized by Mochly-Rosen and co-workers (reviewed in Refs. 9 -11). RACK1 has sequence homology with the ␤ subunit of heterotrimeric G proteins. RACK1 and G␤ are both members of an ancient family of regulatory proteins made up of highly conserved repeating units usually ending with Trp-Asp (WD) (reviewed in Refs. 12 and 13). WD repeat proteins are functionally diverse, although all seem to be regulatory and few are enzymes. The WD repeats in RACK1 are conserved from Chlamydomonas to human (reviewed in Ref. 13). Thus, the function of RACK1 was probably fixed before the evolutionary divergence of plants and ani...