Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast

Abstract

Ark1p (actin regulating kinase 1) was identified as a yeast protein that binds to Sla2p, an evolutionarily conserved cortical actin cytoskeleton protein. Ark1p and a second yeast protein, Prk1p, contain NH2-terminal kinase domains that are 70% identical. Together with six other putative kinases from a number of organisms, these proteins define a new protein kinase family that we have named the Ark family. Lack of both Ark1p and Prk1p resulted in the formation of large cytoplasmic actin clumps and severe defects in cell growth. These defects were rescued by wild-type, but not by kinase-dead versions of the proteins. Elevated levels of either Ark1p or Prk1p caused a number of actin and cell morphological defects that were not observed when the kinase-dead versions were overexpressed instead. Ark1p and Prk1p were shown to localize to actin cortical patches, making these two kinases the first signaling proteins demonstrated to be patch components. These results suggest that Ark1p and Prk1p may be downstream effectors of signaling pathways that control actin patch organization and function. Furthermore, results of double-mutant analyses suggest that Ark1p and Prk1p function in overlapping but distinct pathways that regulate the cortical actin cytoskeleton.

Figure 1

Sla2p-Ark1p two-hybrid interactions and sequence comparisons of kinase domains similar to that of Ark1p. (A) The COOH terminus of Sla2p (residues 581– 968) interacts with residues 380–553 of Ark1p in a two-hybrid assay. The kinase domain of Ark1p is 70% identical to the kinase domain of Prk1p. However, the tails lack significant similarity except for a small motif PxPPPKP, found near the COOH termini of both proteins, once in Prk1p and twice in Ark1p. (B) An alignment of the Ark1p and Prk1p kinase domains with closely related serine-threonine kinase domains from a number of organisms. All are found at the NH₂ terminus of their respective proteins. Thus, the numbering at the side of the alignment corresponds to the residue number within the proteins, except for the S. pombe kinase, for which, in the interests of space, 13 residues (n) have been omitted after the initial methionine. Residues conserved in at least six of the eight proteins are shaded. 12 subdomains are indicated by Roman numerals and these correspond to those defined by Hardie and Hanks (1995). Below the subdomain delineators there is a consensus line which indicates residues conserved in an alignment of 400 kinases. The symbolism used here is the same as that used in Hardie and Hanks (1995): uppercase letters, invariant residues; lowercase letters, nearly invariant residues; o, positions occupied by nonpolar residues; *, positions occupied by polar residues; and +, positions occupied by residues with short side chains and with near neutral polarity. The so-called p-loop domain, involved in binding the nontransferable phosphates of ATP, is indicated by a dashed line. A bullet denotes an invariant lysine residue required for kinase activity (for review see Hanks et al., 1988). After the alignment was made, it was imported into SeqVu (Garvan Institute) for presentation and was annotated in Freehand (Aldus Corp.). (C) An unrooted phylogenetic tree showing how the Ark1p and the Prk1p kinase domains relate to representative kinases from other serine-threonine kinase families. Horizontal bars indicate evolutionary distances between the kinases. The length corresponding to 20% difference in identity is indicated. Note that >100% difference in identity is possible due to the correction for multiple substitutions, which tends to lengthen long branches. The Ark1p and Prk1p kinase domains, together with the kinase domains shown in B, form a newly identified family which we term the Ark family. GenBank accession numbers for the sequences are as follows: Ark1p: 1301849; Prk1p: 763251; YBR059c (S. cerevisiae): 585348; Spo kin (S. pombe kinase): 2894277; Cel kin (C. elegans kinase): 1066951; Ath kin (A. thaliana kinase): 2702277; Hs GAK (Homo sapiens GAK): 2506080; Rn GAK (Rattus norvegicus GAK): 2829846; YPK2: 140977; cAPKa: 125205; PKCa: 125549; AKT1: 400112; S6K: 125695; bARK1: 114151; PVPK1: 125568; DBF2: 1706307; SPK1: 134835; KIN1: 2507199; caMYII: 125285; CKIIa: 125257; MCK1: 126820; ERK1: 232067; SGV1: 134474; and CDC2Hs: 115922.

Figure 2

Phenotypes of single deletions of ARK1 and PRK1, and of ark1Δ prk1Δ double-mutant yeast. Deletions in the ARK1 and PRK1 genes were created as described in Materials and Methods. All strains were grown to log-phase in rich liquid medium (YPD) at 30°C before fixation and visualization. (A–C) DDY130 (wild-type); (D–F) DDY1558 (prk1Δ:: LEU2); (G–I) DDY1407 (ark1Δ::HIS3); (J–L) DDY1564 (ark1Δ::HIS3, prk1Δ:: LEU2). (A, D, G, and J) Actin localization, visualized by rhodamine-phalloidin staining; (B, E, H, and K) nuclear and mitochondrial DNA localization visualized by DAPI staining; (C, F, I, and L) DIC images. Bar, 5 μm.

Figure 3

Actin clumps found in ark1Δ prk1Δ double mutants also contain Sac6p, cofilin, and Sla2p. ark1Δ prk1Δ double mutants were fixed and stained for actin (detected by FITC-conjugated secondary antibody) and for either Sac6p, cofilin, or Sla2p (detected by CY3-conjugated secondary antibody). (A–C) Actin localization in ark1Δ prk1Δ double mutants. (D) Sac6p localization, (E) cofilin localization, and (F) Sla2p localization in the same cells shown in A–C. Bar, 5 μm.

Figure 4

A stereo-pair showing a three-dimensional reconstruction of ark1Δ prk1Δ double-mutant yeast in which the actin has been labeled using rhodamine-phalloidin. The three-dimensional reconstruction from confocal serial sections was produced using Imageworks software running on a Silicon Graphics workstation.

Figure 5

Prk1p and Ark1p localize to cortical actin patches. (A) Indirect immunofluorescence of myc-tagged Prk1p and actin in prk1Δ cells. Epitope-tagged Prk1p colocalizes with cortical actin patches. The control cells show ark1Δ prk1Δ cells bearing vector with no insert, stained with the same combination of antibodies. (B) Indirect immunofluorescence of GFP-Ark1p and actin in wild-type cells. Cortical patches containing actin were found also to contain GFP-Ark1p. Two cells are not expressing Ark1p-GFP. They may be dead or they may have lost the plasmid. Cortical actin patches and actin bars occasionally survive after the death of the cells. (C) Indirect immunofluorescence of GFP-Prk1p and actin in wild-type cells. GFP-Prk1p is found to be associated with actin patches. The central cell shows a strongly staining actin bar, a feature often seen in cells that are dead or dying. This cell is dead since there is no longer any GFP fluorescence. An actin bar is also visible in the left-hand cell, but this cell is still alive and expressing GFP-Prk1p. GFP-Ark1p or GFP-Prk1p overexpression leads to delocalization of the cortical patches and actin bars. Bar, 5 μm.

Figure 6

Effects of Ark1p and Prk1p overexpression. Elevated levels of either Ark1p or Prk1p result in a number of cellular abnormalities. These abnormalities include unusual bud morphologies, septation defects and multiple buds, and death. (A) Rhodamine-phalloidin (i) and DIC microscopy (ii) of selected fields from cultures overexpressing GFP-Prk1p (DDY130 transformed with pDD554) at 25°C for 8 h. Similar results were obtained when native Prk1p was overexpressed (data not shown). Cells that display abnormal internal morphology and that do not stain for actin are indicated by arrowheads. These cells are most likely dead. Continued overexpression of Prk1p ultimately leads to cell death in a majority of the cells. (B) Rhodamine-phalloidin (i) and DIC microscopy (ii) of selected fields from cultures overexpressing GFP-Ark1p (DDY130 transformed with pDD555) at 25°C for 8 h. (C) Cells with abnormal, brain-like, intracellular morphologies are dead. (i) DIC microscopy and (ii) FUN-1 staining of living and dead cells overexpressing Prk1p. In living cells, the FUN-1 is internalized to the vacuole where it is processed into an aggregate that fluoresces brightly (as in the two cells on the right-hand side). Dead cells display a diffuse pattern of fluorescence throughout the cell (as in the cell on the left-hand side).

Figure 7

(A) Dilution series of wild-type (DDY130) cells expressing various GFP fusion proteins under the control of the GAL1,10 promoter. (i) GFP-Prk1p (pDD554); (ii) Ark1p-GFP (pDD555); (iii) GFP alone (control; pTS408); (iv) GFP-Prk1p_(K56A) (pDD561). Cells were grown on glucose-containing medium before dilution and subsequent growth on galactose-containing medium to induce overexpression of the constructs. The plate was photographed after 4 d of growth. (B) (i) Rhodamine-phalloidin staining of filamentous actin and (ii) GFP localization in DDY130 cells overexpressing Ark1p_(K56A)-GFP. The actin patches are polarized normally. Ark1p_(K56A)-GFP localizes to cortical actin patches, but not to actin cables (arrowhead). Bar, 5 μm.

Figure 8

Ark1p-GFP and Prk1p-GFP localization to cortical patches is reduced in abp1Δ cells, but not in sla2Δ, sac6Δ, srv2Δ, or rvs167Δ cells. (A) Actin and (B) Prk1p-GFP localization in (i) sac6Δ, (ii) abp1Δ, (iii) rvs167Δ, (iv) srv2Δ, and (v) sla2Δ cells. (C) Actin and (D) Ark1p-GFP localization in (i) sac6Δ, (ii) abp1Δ, (iii) rvs167Δ, (iv) srv2Δ, and (v) sla2Δ cells. sac6Δ (DDY318), abp1Δ (DDY322), rvs167Δ (DDY950), srv2Δ (DDY952), or sla2Δ (DDY1166) cells were transformed with either pDD554 (GFP-Prk1p) or pDD555 (GFP-Ark1p), expression was induced for 8–12 h, and actin and GFP localization was examined. GFP-Ark1p and GFP-Prk1p are mostly found in the cytoplasm when expressed in abp1Δ cells. In only a small population of cells (<10%) can faint cortical patches of GFP-Ark1p or GFP-Prk1p be detected. Bar, 5 μm.

Figure 9

(A) A summary of the physical and synthetic genetic interactions between the proteins investigated in this study. (B) A potential signal-flow diagram for actin regulation involving Ark1p and Prk1p in budding yeast.

Figure 9

(A) A summary of the physical and synthetic genetic interactions between the proteins investigated in this study. (B) A potential signal-flow diagram for actin regulation involving Ark1p and Prk1p in budding yeast.