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. 2004 Dec 15;18(24):3117–3130. doi: 10.1101/gad.314904

Figure 2.

Figure 2.

Phosphorylation of Crk1 T-loop is required for in vivo activity. (A) crk1 and crk1AEF both fused to myc epitope under the control of Pcrg1 and are shown schematically. (Top) Sequences surrounding the TEY motif and the WYRAPE motif found in kinase subdomain VIII from Crk1 are shown. (B) Phosphorylation of T-loop of Crk1. Extracts were prepared from UME61 (FB1 Δcrk1 Pcrg:crk1-myc) and UME62 (FB1 Δcrk1 Pcrg:crk1AEF-myc) cultures grown in inducing conditions (CMA, complete medium with 1% arabinose) or repressive conditions (CMD, complete medium with 1% glucose) at OD600 of 0.5. The same blot, after stripping steps, was incubated with anti-pTEpY to detect T-loop phosphorylation and with anti-myc to detect myc-tagged proteins. As an internal loading control, we used anti-PSTAIRE to detect Cdk1. (C) The T-loop phosphorylation is required for successful mating on charcoal plates. FB1, UMP12 (FB1 Δcrk1), UME61 (FB1 Δcrk1 Pcrg:crk1-myc), and UME62 (FB1 Δcrk1 Pcrg:crk1AEF-myc) cells were cospotted with FB2 cells in charcoal-complete medium plates with glucose (CMD charcoal) or arabinose (CMA charcoal) as carbon source. (D) Requirement of T-loop phosphorylation for Crk1-mediated hyperpolarized growth. UMP12 (FB1 Δcrk1), UME61 (FB1 Δcrk1 Pcrg:crk1-myc), and UME62 (FB1 Δcrk1 Pcrg:crk1AEF-myc) cells were incubated in CMD and CMA for 6 h. Bar, 10 μm.